the bioenergetics of paracoccus denitrificans

Biochimica et Biophysica Acta, 463 (1977) 129-153 © Elsevier/North-Holland Biomedical Press

BBA86040

T H E B I O E N E R G E T I C S O F P A R A C O C C U S DENITRIFICANS*

P. JOHN and F. R. WHATLEY

Botany School, University of Oxford, South Parks Road, Oxford OXI 3RA (U.K.)

(Received September 27th, 1976)

CONTENTS

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129

II. Nutritional adaptability . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131

III. Mitochondrial features . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133

IV. Oxidative phosphorylation . . . . . . . . . . . . . . . . . . . . . . . . . . . 136

A. Oxidative phosphorylation in membrane particles . . . . . . . . . . . . . . . 137

B. H+: O ratios . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 139

C. Adenine nueleotide changes in intact cells . . . . . . . . . . . . . . . . . . . 141

D. Molar growth yields . . . . . . . . . . . . . . . . . . . . . . . . . . . . 142

E. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143

V. ATPase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 144

VI. Membrane transport . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 146

VII. Cytochrome c-550 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 150

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 151

I. INTRODUCTION

Paracoccus denitrificans is a b a c t e r i u m wh ich c o m m a n d s o u r a t t e n t i o n o n t w o

c o u n t s : its g rea t n u t r i t i o n a l adap t ab i l i t y a n d its r e m a r k a b l e m i t o c h o n d r i a l affinity.

I t was its ab i l i ty to use n i t r a t e as an a l t e rna t ive t e r m i n a l e l ec t ron a e c e p t o r to o x y g e n

wh ich first a t t r a c t e d s tudy o f P. denitrifieans, b u t it s o o n b e c a m e a p p a r e n t t ha t this

versa t i l i ty was on ly o n e e x a m p l e f r o m a wide va r i e ty o f nu t r i t i ona l op t i ons o p e n to

P. denitrificans. K I u y v e r [2] j u s t i fy ing an e legan t a c c o u n t o f t he w o r k o f his g r o u p in

the ear ly 1950s, w r o t e t h a t " I h a v e dwe l t at s o m e l eng th on the specia l case o f M. deni-

Abbreviations: FCCP, carbonylcyanide p-trifluoromethoxy-phenylhydrazone; TMPD, N, N, N' , N'- tetramethylphenylenediamine.

* Previously Micrococcus denitrificans [1].

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trificans because it seemed to me that this might be an effective way of impressing upon the reader the marvel of the characteristic of life that is embodied in the word flexibility".

When studies of the physiology of whole cells gave way in the following decade to studies of the components of the respiratory chain P. denitrificans was shown to possess a constitutive, aerobic respiratory chain similar to the mitochondrial respiratory chain, and different from the respiratory chains of other bacteria which had by then been studied in comparable detail e.g. Escherichia coli, Azotobacter vinelandii, and Mycobacterium phlei. From a comparison of P. denitrificans with the mitochondrion we suggested that P. denitrificans may be thought of as a free-living, highly adaptable mitochondrion. This suggestion was based on the conclusion that oxidative phosphorylation and the constitutive respiratory chain in P. denitrificans resemble oxidative phosphorylation and the respiratory chain in mitochondria. The differences between P. denitrificans and mitochondria, such as the presence of a respiratory nitrate reductase and the absence of an adenine nucleotide carrier in P. denitrificans, could be explained as adaptations to the different environments of the free-living bacterium and of the subcellular organelle.

Inevitably, this view led to P. denitrificans being cast in the role of mitochondrial ancestor, within the general framework of the endosymbiotic theory of the evolutionary origin of the eukaryotic cell [3,4]. A hypothetical transition from the plasma membrane of P. denitrificans to the inner mitochondria membrane was demonstrated to be both feasible and simple. This transition would require: (1) the retention of the constitutive respiratory chain and oxidative phosphorylation, (2) a loss of the adaptive components of the respiratory chain, (3) a modification in the mode of operation of the carrier systems, and (4) the acquisition of only one entirely new component, the adenine nucleotide carrier.

Evolutionary hypotheses are necessarily more speculative than non-evolutionary hypotheses, and evolutionary hypotheses at the cellular level are particularly immune from experimental testing. However, the formulation of a reasonably acceptable hypothesis for the evolutionary origin of the mitochondrion could provide an historical perspective of value in our understanding of the role of the mitochondrion in the cell.

Aside from evolutionary considerations, a comparison of the bioenergetics of the inner mitochondrial membrane with that of the plasma membrane of P. denitrificans is an interesting exercise. Both types of membrane conserve the free energy released by the flow of reducing equivalents along similar respiratory chains from NADH and succinate to oxygen. In mitochondria oxidative phosphorylation is of paramount importance, except where heat production has priority [5,6]. In bacteria the plasma membrane supports oxidative phosphorylation but it also defines the metabolic boundary of the cell and hence is additionally responsible for the uptake of substrates, and for the maintenance of an appropriate internal ionic environment.

We have already made a detailed comparison of P. denitrificans with a mitochondrion [7]. Although that review article was submitted in 1974, publication has

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been delayed until early 1977 due to circumstances beyond the authors' control. A summary of this comparison and its evolutionary implications have already been published in a full paper [8] and in a shortened form [9]. Since these articles were written: (1) new data have appeared on oxidative phosphorylation and membrane transport in P. denitrificans; and (2) the cytochrome c-550 of P. denitrificans has been sequenced and the homology of this cytochrome with mitochondrial cytochrome c and the recently sequenced cytochromes c2 of a variety of photosynthetic bacteria has become apparent. The aim of the pre~nt review is to outline our knowledge of P. denitrificans and to comment on these recent developments from the viewpoint of the already established adaptability and mitochondrial affinity of P. denitrificans.

II. NUTRITIONAL ADAPTABILITY

P. denitrificans can grow either autotrophically with hydrogen and carbon dioxide or heterotrophically with a wide variety of carbon compounds. It can grow aerobically or it can adapt to grow anaerobically if nitrate, nitrite or nitrous oxide are available as terminal electron acceptors [2].

In the absence of an organic source of carbon, P. denitrificans can use hydrogen as reductant and it can fix carbon dioxide by the operation of the reductive pentose phosphate cycle [10]. When methanol or formate is supplied as the sole carbon and energy sources it is oxidized to carbon dioxide, which is again fixed via the reductive pentose cycle [11,12]. It has been pointed out [12] that the incorporation of the carbon of methanol by the fixation of carbon dioxide involves a greater expenditure of energy than if methanol were to be incorporated directly as in other bacteria. The advantage gained by P. denitrificans in using this energetically less efficient mode of metabolism appears to be that it gains a capability for growing on one-carbon compounds with the minimum elaboration of new biosynthetic enzymes [12].

Under heterotrophic conditions P. denitrificans is capable of utilizing a wide variety of compounds as sources of carbon and energy [13]. An indication of this versatility is provided by the finding [1] that of a total of 143 different organic compounds offered, 64 could be utilized as the sole source of carbon.

Enzyme assays of cell-free extracts ofP. denitrificans have revealed the pathways by which some of these compounds are dealt with. Glucose is metabolised via the Entner-Doudoroff pathway or by the hexose monophosphate pathway or by a combination of these two pathways. The glycolytic pathway is absent [14,15]. Pyruvate, succinate and malate are oxidized to carbon dioxide via the tricarboxylic acid cycle, which has been shown to operate in P. denitrificans [16]. When acetate is the sole carbon and energy source it is metabolised by the glyoxylate cycle [10]. When glycollate is the sole carbon and energy source the fl-hydroxyaspartate pathway operates. This pathway is apparently unique to P. denitrificans [17]. It effects the direct condensation of two 2C compounds to a 4C dicarboxylic acid.

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Cells + I mln

1.4 13 ~ 11 ~ugatoms humol

0.0 - 0 .I. ~ 3 ' N ^ - , \ , ~ . _ 0 . 0 0 . 0

_ r~ ~ r~ - - - ~ ' , . ! '~ ; \ \1.9 ' \

, ,, i\ N_L

L-- .... ,t.___ J • . . . . ~_.

t t t H2% H2% %%

Fig. 1. Reduction of oxygen and nitrate by intact P. denitrificans. The bacteria were grown with nitrate as the terminal electron acceptor. Cells (96 mg dry wt) were added to a reaction mixture (50 ml) containing 200 mM sucrose, 20 mM Tris • HC1 (pH 8.0), 10 mM sodium succinate, 5 mM NaNO3 and 5 /~1 of catalase. Temperature, 30°C. Oxygen and nitrate reduction were followed simultaneously by the use of electrodes. The rates of reduction given alongside the traces are expressed in #gatoms oxygen (- - - -) and in/~moles nitrate ( - - - - ) on a per s/g dry wt basis. Modified from John [21].

P. denitril~cans cannot ferment i.e. it cannot use an organic compound as a terminal electron acceptor. Thus anaerobic growth is strictly dependent upon the presence of nitrate, nitrite or nitrous oxide [2]. When nitrate is added as the terminal electron acceptor it is reduced to gaseous nitrogen via nitrite and nitrous oxide. Cells grown with adequate aeration in the presence of nitrate are unable to use nitrate as a terminal electron acceptor, since the synthesis of the respiratory nitrate reductase is repressed by oxygen [18]. However, cells grown anaerobically with nitrate are still able to use oxygen, since the oxidase is a constitutive feature of the cell [2,19]. When cells which have adapted to use nitrate are provided with both nitrate and oxygen they use oxygen in preference to nitrate; only when oxygen is not available do they use nitrate as a terminal electron acceptor [20]. A clear demonstration of this preference for oxygen is provided in Fig. 1, which shows that nitrate respiration proceeds only when the cells have used up all the oxygen in the medium. The nature of the control mechanism by which reducing equivalents are directed to oxygen rather than to nitrate is unknown, but it is pertinent to note that this control is inoperative in respiratory vesicles derived from the plasma membrane of P. denitrificans, which reduce nitrate and oxygen simultaneously and at similar rates (Fig. 2). The possible nature of the switching mechanism which in cells of P. denitrificans (and of E. coli) preferentially directs reducing equivalents towards oxygen is discussed elsewhere [21 ].

Respiratory nitrate and nitrite reduction have also been reported for P. denitrificans cells encapsulated within hydrophobic liquid membranes in which a synthetic anion-transport facilitator is incorporated [22]. Microencapsulated cells reduce

133

M e m b r a n e

vesic les

lmin

- ~ 9.3 'I' 1; /uga toms pmol

. . . . . . 1

1 9" , , ^ -

t H202

Fig. 2. Reduction of oxygen and nitrate by membrane vesicles ofP. denitrificans. Membrane vesicles were prepared from cells grown with nitrate as the terminal electron acceptor. Vesicles (3.5 mg protein) were added to a reaction mixture (50 ml) which contained 20 mM Tris • HC1 (pH 8.0), 30 mM ammonium acetate, 0.5/zg gramicidin D, 2 mg of alcohol dehydrogenase (about 800 units), 0.6 ml ethanol, 0.6 mM NAD ÷, 5 mM NAN03 and 5 #1 catalase. Temperature, 30°C. The rates of reduction given alongside the traces are expressed in /~gatoms oxygen ( - - - - ) and #mol nitrate, ( ) on a per min/mg protein basis. From John [21].

nitrate and nitrite over a wider pH range than free-living cells, they have a decreased

sensitivity to inhibitors, and they are also viable for longer periods.

Denitrifying bacteria, like P. denitrificans, are increasingly being employed in

the purification of potable water supplies [23]. Effluents which emerge from sewage treatment plants or which drain from chemically fertilised farmland can contain

unacceptably high levels of nitrate. By holding these effluents under anaerobic conditions bacterial denitrification is encouraged. As a result of this, the nitrate is removed as nitrogen gas.

III. MITOCHONDRIAL FEATURES

Table I presents in a summarised form the mitochondrial features which have

been identified in the plasma membrane of P. denitrifieans. This table also indicates briefly the distribution of these features among other facultatively or obligately aerobic bacteria. The nature of the evidence for most of these features is discussed in detail elsewhere [7]. The distribution of particular mitochondrial features among the different bacterial species can be gleaned from pertinent recent reviews which have appeared on bacterial respiratory chains [25], cytochromes [26], cytochrome oxidases [27], oxidative phosphorylation [28] and ATPases [29,30].

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TABLE I

MITOCHONDRIAL FEATURES IDENTIFIED IN P. DEN1TR1FICANS

From John and Whatley [7,8] with additional information from Harris et al. [24]* and John, P. and Ferguson, S. J. (unpublished)**

Mitochondrial features of P. denitrificans

Respiratory chain Succinate and NADH dehydrogenases, transhydrogenase, FeS proteins 2 b-type and 2 e-type cytochromes easily distinguishable by difference spectrophotometry

Ubiquinone-10 as sole quinone

Cytochrome aaa as oxidase

Sensitivity to low concentrations of antimycin NADH oxidation inhibited by rotenone and piericidin A

Succinate oxidation inhibited by carboxin and thenoyltrifluoroacetone

Oxidative phosphorylation H + : O ratio of 8 with NADPH Respiratory control released by ADP or uncouplers ATPase with tightly bound nucleotides exchangeable on energisation* ATPase inhibited by venturicidin and 7-chloro-4-nitrobenzo-2-oxa-1,3 diazole ATP synthesis, but not ATP hydrolysis, inhibited by aurovertin**

Membrane phospholipids Phosphatidyl choline as a major constituent All fatty acids straight chain saturated and unsaturated

Occurrence in other aerobic bacteria

Ubiquitous

Common among bacteria with cytochrome aaa as oxidase Uncommon: ubiquinone 10 or 8 in Gram negative; naphthoquinone in Gram positive bacteria Common, but many bacteria have cytochromes o, al or d instead Uncommon Inhibition by rotenone rare. Piericidin A not widely tested Not widely tested

Uncommon, usually lower Rarely observed, and always weaker Not widely tested, presumably ubiquitous Not widely tested

Not widely tested

Uncommon Common, but branched chain and cyclopropane fatty acids in many bacteria

Our in ten t ion in presenting Table I is to indicate the overall similarity between

the plasma membrane o fP . denitrificans and the inner mitochondria l membrane. The

possession of individual features in c o m m o n may not be of great significance. In

some cases the simplifications involved in the construct ion of this table may be

construed as oversimplifications. For example, we have noted the 2 b-type cytochromes

can be readily identified by reduced minus oxidized difference spectrophotometry

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in both mitochondria and in P. denitrificans. We have contrasted this feature with the ready identification by this technique of only one b-type cytochrome in some other bacteria such as E. eoli. However, it is known that: (1) the positions of the absorption maxima measured at 77K of the 2 b-type cytochromes are at 558-559 nm and at 562 nm in mitochondria [31], but at 556 nm and at 562 nm in P. denitrificans [32], (2) mitochondria of higher plants and algae contain an additional b-type cytochrome [33,34] and (3) redox potentiometry [35] and fourth order difference spectrophotometry [36] have allowed the resolution of at least two b-type cytochromes in the aerobic respiratory chain of E. coli. Comparisons of inhibitor sensitivities may also be too briefly reported. Antimycin is an example. When increasing concentrations of antimycin are added to beef heart submitochondrial particles oxidizing either succina e or NADH, a sigmoidal titration curve is obtained; 50 ~ inhibition of either NADH- or succinate-dependent respiration is observed with 0.4 nmol antimycin/mg protein [37]. Similarly, when increasing concentrations of antimycin are added to membrane particles of P. denitrificans oxidizing NADH in the presence of an uncoupler, a sigmoidal titration curve is obtained and 50 ~ inhibition is observed at 0.33 nmol antimycin/mg protein (John, P., unpublished). However, compared to NADH oxidation, succinate oxidation by the P. denitrificans particles is relatively insensitive to antimycin [38,39]. Respiratory particles from many other bacteria, for example, E. coli, Bacillus subtilis and A. vinelandii are either insensitive to antimycin or are inhibited only at concentrations much greater than those effective with mitochondrial and P. denitrificans particles [40]. However, studies with membrane preparations from aerobically grown Rhodopseudomonas capsulata [41] suggest that in some bacterial preparations which are partially sensitive to antimycin or require high concentrations for nearly complete inhibition, branching of the respiratory chain [42] may disguise the presence of a high affinity binding site for antimycin which is clearly demonstrable in mitochondria and in P. denitrificans.

The point which we wish to emphasize is that while none of the mitochondrial features shown by P. denitrificans is unique to P. denitrificans, no other species of bacterium so far investigated is known to possess as many. P. denitrificans effectively assembles within a single organism those features of the mitochondrial respiratory chain and oxidative phosphorylation which are otherwise distributed at random among other aerobic bacteria. Moreover, there appears to be no significant feature of the mitochondrial respiratory chain or ATPase which is present in another bacterium, but which is known to be absent from P. denitrificans [8].

The precise extent to which particular features in P. denitrificans resemble the corresponding features in the mitochondrion is difficult to evaluate with our present relatively limited knowledge of P. denitrificans. On the one hand, P. denitrificans ubiquinone-10 and mitochondrial ubiquinone-10 are identicalchemically. Similarities include their presence in similar amounts in both respiratory chains [7,43], their kinetics of reduction [43], and their function [44] in the cytochrome b regions of the two respiratory chains as suggested by the similar antimycin sensitivity of both respiratory chains. On the other hand, the soluble cytochromes c show some differ-

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ences. While P. denitrificans cytochrome c-550 resembles mitochondrial cytochrome c spectrophotometrically and potentiometrically, P. denitrificans cytochrome c-550 is a much larger molecule (by about 30 residues), it has an acidic rather than alkaline isoelectric point, and it has a limited ability to react with mitochorldrial cytochrome oxidase (refs. 45 and 45a; see also ref. 46).

A remarkable similarity between P. denitrificans and the inner mitochondrial membrane is suggested by the reported cross-reactivity of human anti-mitochondrial antibodies with membrane vesicles of P. denitrificans [47]. It will be of interest to see whether this cross-reactivity is confined to P. denitrificans or is a feature of other bacteria.

Structurally, cells of P. denitrificans show no greater similarity to a mitochondrion than do many other bacterial cells. P. denitrificans is a small immotile short rod or coccus of about 1 / ,m diameter. Electron micrographs of thin sections [7, 48, 49] reveal a layered structure to the cell wall characteristic of gram-negative bacteria, and a plasma membrane which is closely applied to the cell wall. Application of the freeze- etching technique [50] has suggested the presence of small rod-like invaginations of the plasma membrane. The significance of these invaginations is not yet known.

IV. OXIDATIVE PHOSPHORYLATION

Oxidative phosphorylation irt a bacterial extract was first demonstrated [51] soon after the discovery of mitochondrial oxidative phosphorylation, but there is still much uncertainty regarding the stoicheiometry of bacterial oxidative phospholyla- tion [52,53]. This is because in intact cells direct measurements of ATP synthesis have proved to be difficult and the P : O ratios of cell-free systems are generally unreasonably low [25,40]. The difficulty with the intact cells lies partly in the im- permeability of the plasma membrane to ADP and ATP (see ref. 7) and partly in the rapidity with which ATP turnover occurs within the cell [54]. For example, in growing ceils of Aerobacter aerogenes the turnover time of the ATP pool has been estimated to be about 2 s [55]. A similar turnover time for the ATP ofP. denitrificans cells can be calculated in the following way. The respiratory rate of P. denitrificans cells supplied with succinate is about 3 #gatoms O/s per g cell dry wt. [56,57]. If we assume that reducing equivalents from succinate enter the respiratory chain predominantly as N A D H due to the operation of the tricarboxylic acid cycle [39] and that the oxidation of N A D H yields a P : O ratio of 2 [58] then the rate of ATP synthesis is about 6 #mol/s per g dry wt. The ATP content of washed P. denitrificans cells is of the order of 10-20 #mol/g dry wt [56]. The ATP pool in resting cells may therefore turn over completely in about 3 s. In growing cells presumably there is a rapid utilisation of ATP coupled to biosynthesis; in non-growing cells the fate of ATP is not known [53,59,60]. In the absence of simple, direct determinations of P : O ratios in intact cells the following four methods have been used for determining bacterial P : O ratios:

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(A) Oxidative phosphorylation in membrane particles; (B) H + : O ratios; (C) Adenine

nucleotide changes in intact cells; (D) Molar growth yields.

Each method has its merits and its shortcomings, and reliance on only one method is unsatisfactory. However, all four methods have now been applied to

P. denitr(ficans and thus an assessment of the stoicheiometry of oxidative phosphorylation in vivo can be attempted for this bacterium.

IVA. Oxidative phosphorylation in membrane particles Table II shows the P : O ratios which have been observed with particles isolated

by a variety of techniques from P. denitrificans cells grown under a variety of con-

ditions. These values provide a lower limit to the P • O ratios of the cells from which the particles are derived, but it is difficult to estimate the extent to which disruption

of the plasma membrane necessary in their preparation has decreased the P : O ratios

observed with the particles. The most faithful reflection of oxidative phosphorylation

in vivo would appear to be observed with the particles described by John and coworkers [8,39,64-67]. These particles are prepared from cells broken by the relatively

gentle procedure of treating ceils with lysozyme to weaken the cell wall, and then exposing the cells to a hypotonic medium. During lysis the plasma membrane prob-

ably stretches a little, bursts, and the membrane fragments reform themselves into vesicles. When cells a~e grown with succinate and nitrate the resulting membrane

TABLE II

P : O RATIOS OF P. DENITRIF1CANS PARTICLES

Growth of cells Preparative P : O ratios Ref. technique

Substrate Terminal NADH Succinate electron acceptor

Succinate 02 sonication 1.02 0.40 61 Succinate NOn- osmotic 1.46 0:48 39

lysis Succinate 02 French press 1.44 0.41 62 Hydrogen 02 French press 0.95 1.00 62 Succinate NOn- osmotic 1.8' - 8

lysis Succinate 02 osmotic 1.21 0.60 58

lysis Glucose 02 osmotic 1.38 0.84 58

lysis Ethanol 02 osmotic 0.79 0.59 58

lysis Propanol 02 osmotic 0.45 0.47 58

lysis

* Measured as ADP : O ratio.

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A

7 / "",h I"

2 / , , ,.o o 0

6.5 7.0 Z5 8.0 8.5

pH

Fig. 3. Effect of pH on respiratory control in P. denitrificans particles. The reaction mixture contained in 3 ml total volume: Tris/phosphate (10 mM in phosphate, pH as indicated), 5 mM magnesium acetate, 30 pl ethanol, 0.3 mg alcohol dehydrogenase (Sigma A7011), 0.6 mM NAD ÷ and membrane particles (0.12 mg protein) [67]. The respiratory rate was measured at 30°C with a Clark- type oxygen electrode before (-- • --) and after the addition of either 0.2 mM ADP (-- © --) or 10 pM FCCP (-- • --). When a combination of 0.3 ~g/ml gramicidin D and 15 mM ammonium acetate was added as uncoupler [64] - - ~ --), the buffer was Tris.HCi (from pH 7.6 to 8.5) and 0.08 mg membrane protein was present. ADP : O ratios ( - - • - - ) were calculated as previously [63].

preparat ion consists of at least 80 % inside-out vesicles [65]. With N A D H as substrate the respiratory rate is increased 2-3-fold by the addit ion of A D P (in the presence

o f P~) and increased up to 10-fold by the addit ion o f an appropriate uncoupler [7,8]. The incleased respiratory rate which is observed in the presence of an uncoupler over that observed during oxidative phosphoryla t ion is not due to an uncoupler- induced

t ransmembrane pH equalisation itself affecting the kinetics of electron t ranspol t (cf. ref. 68) but rather to a de-energisation of the membrane (a true uncoupling) since this additional uncoupler- induced respiratory stimulation is observed over a wide pH range (Fig. 3). The respiration rate on addit ion of A D P appears to be due to a limited capacity o f the ATPase to utilize the energy provided by respiration. Thus there is a significant restraint imposed on respiration even during ATP synthesis. I f the particles were endowed with respiratory capacity in excess o f phosphorylat ing capacity then one might expect that decreasing the rate of respiration would not necessarily reduce the rate of phosphorylat ion. However, it has been observed [67] that decreasing the respiratory rate by addit ion o f rotenone does not significantly alter tbe A D P : O

ratios so that the rates o f ATP synthesis and of respiration fall in parallel.

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The P : O ratios of the tightly coupled particles of P. denitrificans approach 2 with NADH as substrate, but the P : O ratios are only about 0.5 with succinate [8,39]. When the P : O ratios observed with NADH and suceinate in the different types of particle are compared (Table II) it is seen that in particles from cells grown with succinate the succinate-dependent P : O ratios are consistently less than 50 ~ of the NADH-dependent P : O ratios but, in particles from cells grown with hydrogen, ethanol or propanol, the succinate-depertdent P : O ratios are similar to the NADH- dependent P : O ratios. Thus it may be that, in addition to the coupled constitutive pathway which is shared with NADH oxidation, cells grown on succinate have available to them a respiratory pathway from succinate to oxygen (or nitrate) which is not coupled to ATP synthesis. The presence of a non-phosphorylating pathway from succinate would account for the decreased sensitivity to antimycin of succinate oxidation compared to NADH oxidation [38,39] and would also account for the much lower respiratory control ratios obtained with succinate than with NADH [7].

The respiratory pathway from succinate to nitrate also appears to be effectively uncoupled, since with succinate as substrate the P : NO3- ratios are less than 0.1 while with NADH as substrate the P : NOa- ratios are about 1.0 [39]. These P : NOa- ratios were obtained with particles isolated from cells grown with succinate as substrate and nitrate as the terminal electron acceptor. Presumably in these cells most of the ATP synthesis which occurs during the reduction of nitrate is coupled to the oxidation of the NADH produced during the metabolism of suceinate by the tricarboxylic acid cycle [16] rather than to the oxidation of succinate itself.

The lack of significant ATP synthesis coupled to the oxidation of reduced TMPD by phosphorylating particles from heterotrophically-grown P. denitrificans cells [39,61,62] has prompted some authors [56,58,61,62] to question whether electron transport from cytochrome c to oxygen in heterotrophically grown cells is coupled to ATP synthesis. The oxidation of reduced TMPD is coupled to ATP synthesis in beef heart submitochondrial particles [69] and in phosphorylating particles from a variety of other aerobic bacteria [70-72]. However the absence of phosphorylation coupled to the oxidation of reduced TMPD by phosphorylating particles of heterotrophically grown P. denitrificans cells is an unreliable indicator of the absence of energy- coupling in the terminal region of the respiratory chain ofP. denitrificans. Firstly the oxidation of reduced TMPD is not coupled to phosphorylation with particles from autotrophically grown P. denitrificans cells, in which the oxidation of reduced cytochrome c is coupled to phosphorylation [62]. Secondly the oxidation of reduced TMPD is not coupled to phosphorylation in phosphorylating particles (inside out vesicles) from cells grown autotrophically or heterotrophically [62], yet the presence of energy coupling in the terminal region of the respiratory chain is indicated by the accumulative uptake of nutrients driven by the oxidation of reduced TMPD by right-side out vesicles prepared from both autotrophically [65] and heterotrophically [73] grown cells.

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IVB. H+/O ratios According to the chemiosmotic theory [74] the respiratory chains of mito-

chondria and bacteria are arranged within the coupling membrane so that the flow of reducing equivalents along the respiratory chain results in an outward, electrogenic translocation of protons from the mitochondrial matrix or bacterial cell. For mitochondria it has been demonstrated that the flow of reducing equivalents across a single coupling site results in the translocation of 2 protons across the inner mitochondrial membrane (75-79, refs. but see also ref. 80). Furthermore it has been observed that the hydrolysis of each molecule of ATP at the mitochondrial ATPase results in the translocation of 2 protons across the inner mitochondrial membrane [81,82]. Thus the H ÷ :O ratios of mitochondria, determined under the conditions specified by Mitchell and Moyle [76] faithfully reflect the P : O ratios obtained by direct measurements of respiration and oxidative phosphorylation. This reasoning has been extended to bacteria.

P. denitrificans was the first bacterium for which an accurate determination of H ÷ : O ratios was made. After a careful investigation of the conditions necessary for obtaining stoicheiometric proton translocation, Scholes and Mitchell [83] determined

the H ÷ : O ratio to be 8.0 ± 0.1, with SCN- as the charge-neutralising anion and with endogenous substrate(s) as the reductant. Scholes and Mitchell [83] proposed that this H ÷ : O ratio was attributable to the operation of a transhydrogenase and a respiratory chain operating in series, both of which have the same proton translocation stoicheiometry shown by the mitochondrial transhydrogenase and respiratory chain, with the translocation of 2 protons per pair of reducing equivalents traversing the transhydrogenase and of 6 protons per pair of reducing equivalents traversing the respiratory chain from NADH to oxygen [84]. In the cells used in these experiments it is assumed that NADPH accumulates anaerobically and that, when an oxygen pulse is introduced, reducing equivalents are drawn from this N A D P H via the transhydrogenase to NADH, and thence via the respiratory chain to oxygen. Thus the effective respiratory chain to oxygen. Thus the effective respiratory reductant in the cells ofP. denitrificans used in the experiments of Scholes and Mitchell [83] is NADPH.

It was subsequently reported [84, 85] that H + : O ratios close to 8 can also be observed in rat liver mitochondria after treatment with N-ethylmaleimide. The N- ethylmaleimide probably inactivates succinate dehydrogenase and NAD+-linked enzymes without affecting the mitochondrial NADP-linked isocitrate dehydrogenase, N A DH oxidase or transhydrogenase [84]. Thus NADPH accumulates under anaerobic conditions and acts as the main respiratory substrate [84, 85]. The N-ethylmaleimide treatment effectively alters the availability of reductants for the mitochondrial respiratory chain, and 'allows' the mitochondrion to give similar H ÷ : O ratios to those observed in cells of P. denitrificans.

Lawford et al. [32] have recently extended the studies of Scholes and Mitchell [57,83] by determining the H ÷ : O ratios of P. denitr(ficans cells starved of endogenous substrates, and supplied with substrates from the suspending medium. Cells grown aerobically with malate as substrate were taken from cultures at the late exponential/

141

early stationary phase of the culture, and then allowed to respire in the absence of added substrate. This procedure results in the cells becoming only partially depleted of endogenous respiratory reserves. P. denitrificans appears to retain its respiratory reserves tenaciously. Thus cells starved by aeration for 5 days "still contain a con- siderable amount of endogenous substrate" [56]. The reserve is generally considered to be poly-fl-hydroxybutyrate [49]. When the starved cells used by Lawford et al. [32] were supplied with succinate the H ÷ : O ratios were between 3 and 4, while with malate the H + : O ratios varied greatly between different batches of cells, around an average of value of 4.6. With malate, respiration was abolished upon addition of piericidin A, an inhibitor of NADH dehydrogenase, but with succinate, respiration was unaffected by piericidin A [32]. Thus it was concluded that in the presence of succinate, NADH oxidation did not contribute to the observed respiration and that the succinate dehydrogenase-oxygen span of the respiratory chain has a limiting H ÷ : O ratio of 4. The variability in the H ÷ : O ratios observed with malate prompted Lawford et al. [32] to determine the effect of the age of the culture on the H + : O ratios of the cells. They found that as the culture aged from a logarithmic to a stationary phase the H ÷ :O ratios decreased from 5.8 to 2.9 with endogenous (unknown) substrates and from 7.9. to 3.5 when malate was supplied. The respiratory rates were unaffected by the age of the culture. This decrease in H + : O ratios was interpreted [32] as indicating a loss in the proton-translocating ability of the transhydrogenase and of the NADH-ubiquinone region (site I) of the respiratory chain. They consideied an alternative explanation that in stationary phase culture the cells oxidise malate through an additional flavoprotein-linked dehydrogenase which does not involve NAD(P) + reduction but communicates directly with the ubiquinone pool, but considered this less likely because of the piericidin-sensitivity of malate oxidation by cells yielding the lower H ÷ : O ratios. Whatever the basis of the changes in H + : O ratios resulting from changes in the growth phase, these results indicate that P. denitrificans is capable of altering the efficiency of respiratory energy coupling in response to changing environmental conditions.

IVC. Adenine nucleotide changes in intact cells

When cells of P. denitrificans are allowed to become anaerobic, cellular ATP and ADP levels are low and the AMP level is high. On introduction of oxygenthe ATP and ADP levels rise rapidly and the AMP level falls equally rapidly. Measure- ment of these changes in the levels of adenine nucleotides on the transition to aerobic conditions has yielded estimates of P : O ratios of 1.0 [58] or 1.8 [56]. Application of a similar technique to cells of Proteus mirabilis, A. aerogenes, Pseudomonas aeruginosa and A. vinelandii has yielded P : O ratios of 0.3-1.0 [86]. The P : O ratios measured in this way have the merit that they are direct and that they are made with intact cells. Nevertheless, the significance of the values obtained for P. denitrificans remains uncertain. Firstly, it is difficult to evaluate the rate at which ATP is utilised during ATP synthesis [86]. If the utilisation is rapid a serious underestimate of the extent of ATP synthesis would result. Secondly, the cells used by van Verseveld and

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Stouthamer [58] were obtained from cultures grown to the "late logarithmic phase" and the P : O ratios may not have indicated the maximum phosphorylating capacity in view of the decrease in the H ÷ : O ratios which are associated with the transition from the logarithmic to the stationary phase [32]. Thirdly, the substrates which actually donate reducing equivalents to the respiratory chain have not been positively identified. The cells were starved before the experiment but presumably accumulated N A DH and NADP H anaerobically. These were the probable donors [58].

Van Verseveld and Stouthamer [58] point out that the P : O ratios measured with intact cells are similar whether succinate or propanol is the growth substrate, yet phosphorylating particles isolated from cells broken by osmotic lysis give higher P : O ratios when succinate is the growth substrate than when propanol is the growth substrate (Table II). The explanation they offered was that the influence of the carbon source in the growth medium is to alter the conformation of the membrane particles in terms of the percentage of inside-out vesicles. Results from our own laboratory fully support the contention that the growth conditions are critically important in the preparation of membrane vesicles from P. denitrificans cells broken by osmotic lysis. Thus Burnell et al. [65] reported that the same preparation procedure yields predominantly right-side-out vesicles when applied to cells grown with hydrogen and carbon dioxide, and predominantly inside-out vesicles when applied to cells grown with succinate and nitrate. The factors responsible for this curious behaviour of the plasma membrane of P. denitrificans upon lysis have yet to be identified.

1 VD. Molar growth yields The principles and practice of using molar growth yield measurements for

determining P : O ratios have been reviewed recently [87]. It involves, essentially, measuring the yield of cell material obtained at the expense of a known amount of substrate and oxygen. The yield of cell material per mol of ATP is determined by growing cells under fermentative conditions, where known metabolic pathways opeiate and hence where the amount of ATP produced per tool of substrate is known. The P : O ratios may then be calculated after allowance is made for the energy (ATP) used for cell maintenance, which does not contribute to an increase in cell dry weight. This method has the advantage over all the other methods for determing P : O ratios in that intact growing cells are used. However, accurate determinations of P : O ratios are difficult to make because of the many uncertainties involved [53,88,89]. An additional problem is present in P. denitrifieans in that a fermentative mode of growth is impossible with this bacterium and thus the yield of cell material per mol of ATP cannot be estimated directly. However, de Kwaadsteniet et al. [90] have derived a mathematical model from work with Aerobaeter aerogenes in which the calculation of the P/O ratio did not depend on the use of data from anaerobic growth experiments. The model yielded a P/O ratio that was very close to that previously obtained for A. aerogenes, making use of fermentation data to determine the yield of cell material per mole of ATP. Extending this mathematical model to growth yield determinations in P. denitr(~eans (which can be grown only

TABLE III

P : O AND H ÷ : O RATIOS DETERMINED FOR P. DENITR1FICANS

Method Electron donor P : O ratio H ÷ : O ratio

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Ref.

Directly, P : O and NADH 1.5-1.8 8, 39 ADP : O with particles succinate 0.5 39, 58,

61, 62 Directly, rapid changes ? NADH 0.8-1.8 56, 58 in adenine nucleotide l eve l s principally in resting cells Molar growth yields NADH principally < 1.7 58

Respiration-driven ? NADPH 8 32, 83 H + transiocation Succinate 3-4 32

"aerobically"), van Verseveld and Stouthamer [58] calculate the P/O ratio to be no

higher than 1.7 for aerobic growth in a chemostat where the substrate, gluconate,

limited growth.

IVE. Conclusions

The P : O ratios and the H + : O ratios which have been obtained with P. denitrificans are listed in Table III . The H + : O ratios determined for P. denitri.ficans are

similar to the H + : O ratios determined for mi tochondr ia oxidising the same sub- ~, trates. By contrast the P : O ratios determined for P. denitrificans are always lower than the P : O ratios determined for mi tochondr ia oxidising the same substrates as

P. denitrificans. For mitochondria , the limiting H + : O ratios determined under the

condit ions o f Mitchell and Moyle [76] are twice the P : O ratios [75]. For bacteria the limiting H ÷ : O ratios, determined under essentially similar conditions to those

specified by Mitchell and Moyle [76] are generally assumed to be equal to twice the

P : O ratios [91-93]. This assumption is valid if the H + : A T P ratio is 2 (as it appears

to be in mitochondria) and if the bacterial ATPase is the sole consumer o f the pro on motive force generated by the p ro ton translocating respiratory chain. Pro ton trans-

location driven by A T P hydrolysis has been observed with particles o f E. coli [94] but the stoicheiometry o f the ATP-dr iven pro ton translocation is not yet known for

bacteria. The bacterial plasma membrane is known to be responsible for the uptake o f substrates into the cell, for the accumulat ion o f K + and for the active exclusion of

Na + f rom the cell [95]. Present evidence suggests that in aerobic bacteria the respira- t ion-dependent proton translocation drives these t ransport processes [96] in addit ion to driving A T P synthesis. In mi tochondr ia it is known that the accumulat ion of Ca z+ via the mitochondrial Ca 2+ carrier can compete successfully with A T P synthesis and

thus lower the P : O ratios [97, 98]. In bacteria the mechanisms of cation t ransport are largely unknown, but it seems reasonable to expect on a simple thermodynamic basis that active t ransport would compete with ATP synthesis for the proton motive force [99].

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It is not yet known how the energy could be partitioned between transport and ATP synthesis on the bacterial plasma membrane. It is possible that the kinetics of the ATPase and of the transport systems are such that no single system monopolises the energy released by respiration. A kinetic restriction of the P. denitrificans ATPase is suggested by the observation that respiratory control in phosphorylating particles of P. denitrificans is only partially released by the addition of ADP but fully released by the addition of uncouplers [8,64]. In this respect these particles resemble mitochondria treated with partially inhibitory concentrations of oligomycin. Respiratory control in mitochondria so treated is only partially released by the addition of ADP, but fully released by the addition of uncouplers; and they have lower P : O ratios than untreated mitochondria [100]. Thus it is possible that the P : O ratios in P. denitrificans are lower than might be expected from the H ÷ : O ratios because of a kinetic restriction in the operation of the ATPase.

A further factor which may limit the P : O ratio in P. denitrificans is the existence of a pH outside the cell more alkaline than that inside the cell. P . denitrificans cells suspended in media at either pH 6 or 8 were reported by Scholes and Mitchell [57] to have an internal pH between pH 7.2. and 7.4. P. denitrificans can grow over a wide pH range, with a pH optimum between pH 8 and 8.5. (John, P., unpublished). The internal pH of cells growing in a strongly alkaline medium is not yet known, but it seems likely that as the external pH is changed a relatively constant internal pH is maintained rather than a relatively constant transmembrane pH gradient. One con- sequence of a transmembrane pH gradient that is more acid inside than outside is that a chemiosmotic mechanism of ATP synthesis would require protons to move from an alkaline to a more acid phase, under the influence of a membrane potential (negative inside). In E. coli cells it has been observed [101] that ATP synthesis driven by an electrogenic efflux of K ÷ via valinomycin down a concentration gradient of K ÷ was detectable only when the external pH was lower by about 3 pH units than the internal pH. Similarly, in mitochondria, ATP synthesis driven by an electrogenic efflux of K ÷ via valinomycin requires a higher ratio of [K ÷ inside]: [K ÷ outide] when the

external pH is raised [102]. Thus the available evidence indicates that the proton-translocating capacity of

the respiratory chain ofP. denitrificans is basically similar to that of the mitochondrial respiratory chain, but is capable of modification in response to changing growth conditions. The stoicheiometry of oxidative phosphorylation is lowerinP, denitrificans than in mitochondria, possibly as a result of the different roles of the bacterial plasma membrane and the inner mitochondrial membrane.

v. ATPase

Recent studies of the ATPase of P. denitrificans [24,66,67] have used the tightly coupled phosphorylating particles prepared from cells broken by osmotic lysis. These particles have a low rate of ATP hydrolysis compared to the rate of ATP

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synthesis. ATP is hydrolysed at about 0.02/~mol/min per mg protein, whereas ATP synthesis coupled to NADH oxidation occurs at about 2 #mol/min per mg protein [24,66, Fig. 3]. This relatively low rate of ATP hydrolysis fails to support an enhancement of 1-anilino-l-naphthalene-8-sulphonate fluorescence [103] although respiration of succinate does support this enhancement [103,104]. The ATPase activity can be stimulated about 2-fold by treatment with trypsin [24] and about 2-fold by treatment with trypsin [24] and about 5-fold by including 150 mM NaHCOa in the reaction medium [66]. The stimulation by trypsin has been observed with other bacterial ATPases [105-108] where it may be due to the destruction of an inhibitor protein, as in mitochondria [109] but there is no other evidence for the occurrence of an inhibitor protein in P. denitrificans. A stimulation induced by NaHCOa of the mitochondrial ATPase has also been observed but the basis of the stimulation is not understood [110].

Not only is the ATPase activity of the P. denitrificans particles low, but it has been claimed that the ATPase acts essentially irreversibly during oxidative phosphorylation [67], since [y-a2p]ATP incubated with particles respiring in state 3 releases a2P l

at a rate less than 2 ~/o of the rate of ATP synthesis [67]. It follows that if the ATPase is essentially irreversible then: (1) respiratory control must operate through a kinetic rather than an equilibrium mechanism, and (2) the phosphate potential and the energised state of the membrane are never in equilibrium and thus it is inappropriate to consider poising the phosphate potential against the energised state of the membrane (currently monitored as a protonmotive force). A fuller discussion of these aspects of the P. denitr(ficans ATPase is given in a recent review [111].

The P. denitrificans ATPase is inhibited by the chemical modifying agent 7- chloro-4-nitrobenzo-2-oxa-l,3-diazole. Inhibition of both ATP hydrolysis (measured in the presence of 150 mM NaHCOa) and ATP synthesis show a similar time course of inhibition, have a similar pH dependence, and in both cases inhibition is relieved by treatment with dithiothreitol [66]. In its sensitivity to this reagent the P. denitrificans ATPase resembles the ATPases of beef heart mitochondria [112] and of chloroplasts [113]. Unlike other ATPase inhibitors the action of 7-chloro-4-nitrobenzo-2-oxa-l,3- diazole can be understood in chemical terms. In the ATPase of beef heart mitochondria its inhibitory action has been ascribed to the selective modification of a single essential tyrosine residue in the F1 component [112]. The role of this tyrosine residue is not yet understood but it is clear from the results obtained with the P. denitrificans particles [66] that this residue functions equally in the synthesis and hydrolysis of ATP.

A further similarity between the P. denitrificans ATPase and the ATPases of mitochondria and chloroplasts is revealed by the finding [24] that the P. denitrificans ATPase has tight binding sites for ATP and ADP. The tightly bound nucleotides are exchangeable only upon energisation of the membrane. Exchange with ATP and ADP supplied in the medium is observed only during coupled respiration. This exchange- ability is decreased in the presence of uncoupling agents or EDTA but is stimulated by the addition of venturicidin, an inhibitor of the P. denitrificans ATPase [8]. The conditions required for the exchange of these tightly bound nucleotides are compatible with a general mechanism [114] for the mode of operation of the ATPase in which

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energy is required for ATP to be released from the ATPase, but not for the actual synthesis of ATP.

VL MEMBRANE TRANSPORT

Respiration-driven uptake into membrane vesicles from P. denitrificans has been described for amino acids [115], sulphate [65], phosphate [116], and lactate [73]. White et al. [I 15] demonstrated the uptake of glycine, alanine, asparagine and glutamine. From the rates of uptake of each amino acid observed in the presence of the other amino acids it appears that only 2 carriers are involved: a glycine/alanine carrier, and an asparagine/glutamine carrier. The curious feature of the vesicles prepared by White et al. [115] is that they failed to take up other amino acids, carbohydrates, or mono- and dicarboxylic acids. Presumably the cells took advantage of the rich medium in which they were cultured, and the cells accumulated many of the metabolites which the derived vesicles failed to take up. Assuming a plasma membrane localisation for the carriers involved we must conclude that the methods of vesicle preparation and assay used by White et al. [115] were unsuitable for demonstrating uptake of any other metabolites, than the 4 amino acids noted. Subsequently, Nichols and Hamilton [73] prepared vesicles by a different technique from that used by White et al. [115] and were able to demonstrate a carrier-mediated uptake of lactate.

The experiments of Burnell et al. [65,116] were designed to determine the mechanism of sulphate and phosphate uptake into vesicles of P. denitrificans. Two types of membrane vesicles were prepared by the same procedure from cells grown under different conditions (see Section IVA above): right-side-out vesicles from cells grown aerobically with hydrogen and carbon dioxide; inside-out vesicles from cells grown with succinate and nitrate. Respiration-driven uptake of sulphate or phosphate was observed with the right-side-out vesicles but not with the inside-out vesicles. How- ever a transient uptake of sulphate or phosphate was observed with either type of vesicle when a pulse of KC1 (in the presence of nigericin) or of NHaCI was applied. Uptake driven either by respiration or by the addition of salts was completely inhibited by the uncoupler FCCP, and furthermore FCCP added during respiration- driven uptake caused a iapid efflux of sulphate or phosphate. The transient uptakes driven by the addition of salts or by respiration were shown to be carrier mediated by their sensitivity to thiol-group reagents. When sulphate in the growth medium was omitted and cysteine added as the sole S source the resulting vesicles failed to take up sulphate but continued to take up phosphate. Thus it was concluded [116] that sulphate and phosphate have different carriers and that cells grown on cysteine lose the sulphate carrier but retain the phosphate carrier.

Two main conclusions emerged from these studies [65,116]: (1) the sulphate and phosphate carriers are reversible and (2) sulphate and phosphate enter the vesicles by an electroneutral proton symport mechanism. The reversibility of the

147

sulphate and phosphate carriers is indicated by the efflux induced by an addition of FCCP, by the transience of the uptake driven by the addition of salts, and by the similarity in the extents of uptake observed with right side out and inside out vesicles upon the addition of salts.

An electroneutral proton symport mechanism is the simplest explanation of sulphate and phosphate uptake into the vesicles. Thus, addition of KC1 (in the presence of nigericin) or of NH4C1 generates a pH gradient (alkaline inside) across the vesicle membrane by removal of protons from the vesicle interior, either by electroneutral exchange for the K + entering down its concentration gradient, or by their combination with NH3 to give NH4 +. However, when the functioning of these carriers in the plasma membrane of the intact cell is taken into consideration a reversible electroneutral proton-symport mechanism appears to be inapplicable. Thus it is known that P. denitrificans can grow in alkaline media. We have argued above (Section IVE) that the internal pH of the cell under these conditions is likely to be less alkaline than that of the medium. If sulphate and phosphate enter the cell via a reversible carrier by an electroneutral proton-symport mechanism then under these conditions uptake will be impossible. Indeed sulphate and phosphate will flow out of the cell in response to the pH gradient (alkaline outside) across the plasma membrane.

A proton symport mechanism would be adequate to drive the accumulative uptake of sulphate and phosphate from an alkaline medium to a more acid cytosol if it were electrogenic [I 17]. Since respiration generates a membrane potential which is negative inside [81 ] an appropriate stoicheiometry would require at least 3 protons to be translocated with each sulphate and at least 3 protons with each HPO42-. A decision on whether sulphate and phosphate transport occur in P. denitrificans by an electroneutral or by an electrogenic mechanism can only come from measuring the proton- anion stoicheiometry during uptake [ 118]. Measurements of the pH changes associated with the uptake of phosphate into the yeast Saccharomyces carlsbergensis have shown that 3 protons accompany the uptake of each H2PO4- [119]. Similarly, measurements of the pH changes associated with the uptake of alanine into E. coli have shown that cells grown with excess alanine take up alanine by a proton symport mechanism with a stoicheiometry of 1 proton/alanine, while mutants which are selected for their ability to grow under conditions of alanine limitation take up alanine with a stoicheiometry of 2 or 4 protons/alanine [120].

VII. CYTOCHROME c-550

The cytochrome c-550 of P. denitrificans resembles mitochondrial cytochrome c and the c-type cytochromes of many other bacteria in that it is a small protein which can be easily isolated and purified. Because of these properties more is known about the cytochrome c-550 ofP. denitrificans than about any other protein of this bacterium.

P. denitrificans cytoehrome c-550 resembles mitochondrial cytochrome c in that both cytochromes have absorption bands in the ieduced form at similar regions of

148

the visible spectrum, and both cytochromes have a midpoint potential at about 250 mV [121,122]. The P. denitrificans cytochrome c-550 differs from mitochondrial cytochrome c in being an acidic protein with an isoelectric point at about pH 4, and in being a larger molecule with a polypeptide chain length of 134 amino acid residues [122,123]. Mitochondrial eytochromes c have an isoelectric point at about pH 10 and a chain length of 103-113 amino acid residues [46]. As expected from its acidic nature amino acid analysis of P. denitrificans cytochrome c-550 reveals a preponderance of acidic over basic amino acids, the ratio of acidic to basic amino acids being about twice the ratio of that in horse heart cytochrome c [122]. It was noted previously [7] that the fungi Aspergillus oryzae and Ustilago sphaerogena had been reported [124] to possess cytochromes c with an acid or neutral isoelectric point. Subsequent, mine precise measurements [125] reveal that Ustilago sphaerogena cytochrome c has an isoelectric point at pH 9.40, and it now seems likely that all mitochondrial cytochromes c are strongly basic [46].

Despite the differences between P. denitrificans cytochrome c-550 and mitochondrial cytochrome c the two cytochromes appear to be interchangeable to some extent with mitochondrial cytochrome oxidase (cytochrome aaa) and interchangeable to a larger extent with the P. denitrificans cytochrome oxidases (cytochromes aaa and cd) [45, 45a, 46, 121, 126-129]. However, interpretation of the data is complicated because in none of these experiments was it established that endogenous cytochromes c or c-550 were absent from the oxidase preparation used. Thus the possibility that the endogenous c-type cytochrome acted as an intermediate cannot be ruled out, although we note that Smith and coworkers [45, 45a] have attempted to render endogenous c-type cytochromes ineffective by treating oxidase preparations with sodium deoxycholate.

These physical and physiological similarities between P. denitrificans cytochrome c-550 and mitochondrial cytochrome c have prompted analysis of the molecular structure of P. denitrificans cytochrome c-550. Thus, recent publications have proposed an amino acid sequence of the P. denitrificans c-550 [123] and a 3-dimensional folding of its polypeptide chain as revealed by X-ray diffraction methods (refs. 130 and 131; Timkovich, R. and Dickerson, R. E., personal communication). Compar- able high resolution (2.5 A) X-ray diffraction studies have been performed on only two c-type cytochromes: mitochondrial cytochrome c from tuna fish [see ref. 46] and cytochrome c2 from Rhodospirillum rubrum [132]. A preliminary, low resolution (4 •) X-ray analysis has also been reported for the cytochtome c-551 from Pseudomo- has aeruginosa [131]. The complete amino acid sequences of cytochromes c from 67 species of eukaryotes are listed by Dickerson and Timkovich in their compiehensive review [46]. Among bacterial c-type cytochromes the work of Ambler has provided us with amino acid sequences for the cytochromes c2 of 6 species of purple non- sulphur photosynthetic bacteria; for the cytochromes c-551 of 5 species of the genus Pseudomonas; and for the cytochrome c-551 of Azotobacter vinelandii (ref. 133, 134; see also ref. 46).

The amino acid sequence analyses reveal extensive homology between the

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P. denitrificans cytochrome c-550 and both mitochondrial cytochrome c and the cytochromes c2 from purple non-sulphur bacteria [46, 123], The principal differences between P. denitrificans cytochrome c-550 and mitochondrial cytochrome c reside in the presence of extra residues in 3 regions of the P. denitrificans molecule and the addition of 15 residues at the carboxy-terminal end ofP. denitrificans cytochrome c-550. The extra residues inserted into the P. denitrificans c-550 occur in the region of residues 20-30, 50-60 and 70-80 of the mitochondrial cytochrome c. X-ray structure analyses [130] reveal that thege extra residues are arranged as loops on the surface of the molecule; the interior of the P. denitrificans cytochrome c-550 molecule is almost identical with that of the mitochondrial cytochrome c molecule. Additional residues in the 50-60 and 70-80 region are also present in cytochrome c2 from Rhodospirillum rubrum, Rhodopseudomonas palustris, Rhodopseudomonas sphaeroides and Rhodo- pseudomonas capsulata; and additional residues in the 20--30 legion are also present in the cytochromes c2 of Rhodopseudomonas sphaeroides and Rhodopseudomonas capsulata [131]. However, the 15-residue carboxy-terminal tail is unique to P. denitrificans c-550. Having noted that none of the residues of this tail are strongly hydrophobic, Timkovich and Dickerson (personal communication) have suggested that it "hangs loose from the back of the molecule in aqueous solution". The function of the tail is unknown.

Recenlly, Ambler et al. [134] have determined the complete amino acid sequence of the cytochromes c2 from the non-sulphur purple bacteria Rhodomicrobium vannielii and Rhodopseudomonas viridis and demonstrated that these sequences can be aligned perfectly with horse cytochrome c when only a single residue of the horse cytochrome c is deleted. These cytochromes c2 have none of the additional residues present in P. denitrificans cytochrome c-550 and in the cytochromes c2 of other non-sulphur purple bacteria, and thus must be regarded as being more similar to mitochondrial cytochrome c than any other known c-type cytochrome of bacterial origin.

The determination of amino acid sequences in mitochondrial cytochromes c has revealed a clear and consistent relationship between sequence homology and phylogenetic relationships inferred from comparative morphology and palaeontology [46]. This close correspondence between sequence-based and classically-based phylo- genies has encouraged the application of sequence analysis for the generation of the phylogenetic trees in otherwise difficult groups of organisms, like the higher plants [135]. However, when amino acid sequences of bacterial species are compared, dissimilar bacteria may be seen to possess similar eytochrome sequences, and similar bacteria different sequences. Thus while A. vinelandii shows no obvious taxonomic affinity with the genus Pseudomonas its cytochrome c-551 sequence falls within the range of variation found among the sequences of cytochromes c-551 isolated from different species of Pseudomonas [133]. Likewise, the P. denitrificans cytochrome c- 550 sequence resembles the Rhodopseudomonas capsulata sequence as closely as does the Rhodopseudomonas sphaeroides sequence [46]. This similarity may be less sur- prising than it first appears since a strain of Rhodopseudomonas sphaeroides has recently been described [135a] which can grow either photosynthetically under

150

anaerobic conditions or heterotrophically using oxygen or nitrate as terminal electron acceptors.

Following the earlier speculation of Anderson [136] on the importance of transfer factors in bacterial evolution, Hedges [137] appears to have been the first author to have questioned whether data derived from sequence analysis can be used to construct phylogenetic trees for bacteria in the same way as for eukaryotes. He noted that plasmids conferring antibiotic resistance can be transferred between distantly related bacteria, and that these plasmids can be integlated into the bacterial chromosome. From this and other evidence he concluded that bacteria can constitute one gene pool from which any species may draw genes as these are required. Recently, as Hedges [137] puts it, there has been a strong evolutionary demand for genes conferring drug resistance. The implication of this view is that whereas phylogenetic trees are appropriate representations of eukaryotic evolution, a reticulate pattern more faithfully represents the evolutionary course of prokaryotic organisms, where small parts of the genome can cross wide taxonomic gaps.

Faced with the anomolous sequence homologies among bacterial c-type cytochromes Ambler [133,134] and latterly Dickerson and Timkovich [46] suggest that phylogenetic trees are inapplicable to bacteria, and again genetic transfer is offered as a possible complicating factor in bacterial phylogeny.

Whatever the underlying evolutionary mechanisms may be, it appears that sequence homologies between bacterial c-type cytochromes are an unreliable guide to bacterial phylogeny. Furthermore, sequence homologies between mitochondrial cytochrome c and particular bacterial c-type cytochromes are equally unreliable guides to the identification of those present-day bacteria which are most closely related to the postulated mitochondrial ancestor. There is, therefore, no reason to suspect that Rhodomicrobium vannielii and Rhodopseudomonas viridis are more closely related to this ancestor than is P. denitrificans.

We continue to be impressed by the large number of features in common between P. denitrificans and the present-day mitochondria (Table I). Consequently, we believe that the present-day P. denitrificans must have many of the attributes of the bacterial ancestor of the mitochondrion. This conclusion does not necessarily require a close evolutionary relationship between P. denitrificans and the mitochondrial ancestor, although this might be expected.

ACKNOWLEDGEMENTS

We are grateful to Professor R. P. Ambler, Professor R. E. Dickerson, Professor L. Smith, Dr. W. A. Hamilton, Dr. K. W. Hanselmann and Dr. S. J. Ferguson for supplying us with copies of their work prior to publication. Thanks are due to the Journal of General Microbiology for their kind permission to reproduce Figs. 1 and 2. Work from our own laboratory cited in this review has been supported by a grant from the Science Research Council.

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the bioenergetics of paracoccus denitrificans

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