the active species of ‘co2’ utilized by reduced ferredoxin: co2 oxidoreductase from clostridium...

Eur. J. Biochem. 55, 11 1 - 11 7 (1975)

The Active Species of ‘C02’ Utilized by Reduced Ferredoxin : C02 Oxidoreductase from Clos tr idium pas teur ianum

Rudolf K. THAUER, Barbara KAUFER, and Georg FUCHS Abteilung fur Biologie, Ruhr-Universitat Bochum

(Received January 13/March 13, 1975)

Reduced ferredoxin : CO, oxidoreductase (CO, reductase) from Clostridium pasteurianum catalyzes the reduction of ‘CO,’ to formate with reduced ferredoxin, an isotopic exchange between ‘CO,’ and formate in the absence of ferredoxin, and the oxidation of formate to ‘CO,’ with oxidized ferredoxin. The active species of ‘COz’, i.e. CO, or HCO, (H2C03), utilized by the enzyme was determined. The method employed for the species identification was that of Cooper et al. (1968). Both ‘CO,’ reduction to formate and the exchange reaction were studied. Data were obtained which are compatible with those expected if CO, is the active species.

The V and the dissociation constant K, of the enzyme . CO, complex in dependence of pH were determined from initial velocity studies of the exchange reaction. V was found to be only slightly affected by pH between 5.5 and 7.5. K, was markedly dependent on pH; the constant increased with decreasing pH from 0.2 mM at pH 7.5 to 3 mM at pH 5.5.

The reduction of ‘COz’ to formate is an important reaction in the metabolism of many anaerobic bacteria [I]. It is involved, for example, in methane formation from ‘CO,’ in methane bacteria [2,3], in acetate formation from ‘CO,’ in Clostridium thermoaceticum and C. acidi-urici [4- 111 and in one-carbon unit synthesis from ‘CO,’ in C. pasteurianum [12- 151.

An understanding of the physiology and of the mechanism of ‘CO,’ reduction to formate requires the identification of the active species of ‘CO,’, i.e. CO, or HCO; (H,C03) utilized by the enzyme catalyzing the ‘CO,’ reduction. The active species has, however, until now not been elucidated.

In this investigation the active species of ‘CO,’ utilized by the enzyme from C. pasteurianum has been determined. In C. pasteurianum formate is syn- thesized from ‘CO,’ with reduced ferredoxin as electron donor [16]. The reaction is catalyzed by

Abbreviation. Fd, ferredoxin from C. pasteurianum. Enzymes. COz reductase or reduced ferredoxin : COz oxido-

reductase (EC 1.2.7. -); carbonic anhydrase (EC 4.2.1.1); deoxyribonuclease (EC 3.1.4.5); ferredoxin hydrogenase (EC 1.12.7.1); lysozyme (EC 3.2.1.17).

Note. No distinctions are made between CO,, HzCO,, HCO;, and C0:- when the symbol ‘COz’ is used.

~-

reduced ferredoxin : CO, oxidoreductase, which appears to be a molybdoenzyme [15,17]. Evidence will be presented indicating that CO, rather than HCO; (or H2C03) is the substrate of the reductase.

The method employed for the species identification is that of Cooper et al. [18]. It is based upon the observation that at temperatures below 20 “C and in the absence of carbonic anhydrase the hydration of CO, is slow. At 0 “C several minutes are required for the reaction to attain equilibrium when the initial reactants are CO, and H 2 0 [19-211.

\I$?,, f‘l51

5 h r 2

C02 + H2O 77 H 2 C O 3 F H C O ; + Hi

k + , FZ 0.0025 s-’; k _ l z 0.93 s-l (calculated from k-values determined at 25 “C [19] using a Qlo (k,+lo, , /k,) of 2.9 [20]); k , , = lo7 s-’; k - 2 = 5 x 10’’ M-’ s-’; k + , / k _ , = 2 x M [19].

MATERIALS AND METHODS

Argon (spezial), hydrogen (spezial), and CO, (Testqualitat) were obtained from Messer Griesheim

Eur. J. Biochem. 55 (1975)

112 The Active Species of ‘COz’ Utilized by COz Reductase

GmbH (Diisseldorf). Sodium [14C]carbonate and sodium [‘4C]formate were purchased from Amersham Buchler GmbH & Co KG (Braunschweig). Enzymes were supplied by Boehringer Mannheim GmbH (Mannheim, Germany), benzyl viologen, Good’s buf- fers [22] and dithioerythritol from Serva (Heidelberg). Monomeric ferredoxin [23] from C. pasteurianum was prepared as described by Mortenson [24].

CO, Reducrase

C. pasteuvianum (ATCC 6013) was grown on the glucose medium described by Lovenberg et al. [25]. Cell-free lysates were prepared by incubating 10 g of frozen cells (wet weight) in 30 ml HzO with 20 mg lysozyme and 2 mg deoxyribonuclease under hydrogen at 35 ”C for 30 min and by then centrifuging at 35000 x g for 15 min at 0 “C. The supernatant contained approximately 0.9 unit (1 unit = 1 pmol/min at 35 “C) of CO, reductase, 50 units of hydrogenase and 30 mg protein per ml. When stored at - 20 “C under strictly anaerobic conditions practically no activity was lost within a week.

For the kinetic studies a partially purified extract was obtained by anaerobically passing 5 ml of the cell-free lysate through a small column (Whatman, 1 cm diameter) which was filled with a 4-cm layer of Dowex-2-acetate (X8, 100-200 mesh) and a 2-cm layer of DEAE-cellulose (DE 52, Whatman). Prior to use the column was equilibrated with 10mM imidazole acetate buffer pH 7.2, containing 35 mM mercaptoethanol. The eluate, which was free ’ of ferredoxin and of all anions except acetate, contained approximately 0.8 unit of COz reductase, 40 units of ferredoxin hydrogenase and 25 mg of protein per ml. The absence of formate and of ‘COz’ was routinely checked with [14C]formate and ‘14C02’ added to the lysates prior to column treatment. When stored under strictly anaerobic conditions at 0 “C the partially purified extract was stable for at least 5 h.

Assaj) Conditions

‘CO,’ reduction to formate with reduced ferredoxin and isotopic exchange between ‘CO,’ and formate in the absence of ferredoxin were followed by measuring the formation of [14C]formate from ‘14COz’. ‘CO,’ reduction to formate was determined using a reduced ferredoxin regenerating system consisting of Hz and ferredoxin hydrogenase. Ferredoxin hydrogenase was present in high activities in the partially purified extracts used in the experiments. It should be noted, however, that the hydrogenase reaction is thermo- dynamically not very favorable (H, + Fd,,, $Fdte;, + 2 H + ; AG6 % 0 kcal/mol). At equilibrium only

approximately 50 % of the ferredoxin (Fd,,,/Fdred, ; Ed = -400 mV [26]) is present in the reduced form at pH 7 and a hydrogen pressure of 1 atm (101 kPa) (H+/H2; Ed = -420 mV). Thus the kinetics of ‘CO,’ reduction to formate had to be determined in the presence of finite amounts of the product oxidized ferredoxin.

Determination of the Active Species of’ ‘CO,’ The assay conditions were essentially those

described by Cooper et al. [18]. The experiments were carried out at 0 “C under strictly anaerobic conditions in 7-ml test tubes (total volume) closed with a rubber stopper. 3-ml assay mixtures were used leaving 1.8-ml gas phase. Anaerobic conditions were obtained by repeatedly evacuating and refilling the test tubes with either argon or hydrogen. The reaction was initiated by injection of enzyme, followed by HCO; and then, in about 6 s, by an identical molar amount of CO,. The COz was prepared from potassium bicarbonate at 0 “C by the addition of 0.1 ml 1 M acetic acid to 1 ml 0.1 M bicarbonate solution 3 min prior to use (at the resulting pH of approximately 5 , 3 min are enough for the reaction to attain equilibrium). Either the CO, or the HCO; was labeled with I4C and the accompanying member of the pair was unlabeled. The addition of the nonradio- active species of ‘COz’ assured that prior to isotopic equilibrium one member of the pair will have a higher specific activity than the other and that the assay conditions are identical in both experiments. The assay mixture was continuously stirred with a small magnetic paddle until 9 s after the COz was added. Then special care was taken to avoid any further mixing in order not to lose CO, into the gas phase. The amount of H2 dissolved when H, was the gas phase was sufficient to secure the continuous regenera- tion of reduced ferredoxin via its hydrogenase. At 0 “C and 1 atm (101 k Pa) pressure 0.96 pmol of H2 are dissolved in 1 ml distilled H 2 0 [27].

Following initiation of the reaction the mixture was sampled at approximately 20-s intervals. 0.2-ml samples were quickly withdrawn with a Hamilton syringe and injected into 0.5 ml 5 “4 trichloroacetic acid and then analyzed for [14C]formate. Detailed assay conditions are given in the legend to Fig. 1.

Determination of Kinetic Constants For ‘CO,’ reduction to formate and isotopic ex-

change between ‘COz’ and formate, the assays were carried out at 35 “C under strictly anaerobic conditions in 7-ml test tubes (total volume) closed with a rubber stopper. 1-ml assay mixtures were used leaving a gas phase of 3.8 ml. Anaerobic conditions were


R. K. Thauer, B. Kaufer, and G. Fuchs 113

obtained by repeatedly evacuating and refilling the tubes with either argon or hydrogen. The liquid and the gas phase in the tubes were continuously equilibrated by rapid shaking. The reaction was started by injection of enzyme solution with a Hamilton syringe and stopped after 1,2 or 5 min by the injection of 0.2 ml 20% trichloroacetic acid. Then the mixture was analyzed for [14C]formate. It was secured that under the experimental conditions the reactions pro- ceeded linearly with time and the rates were propor- tional to the amount of enzyme added. Thus it can be assumed that steady-state conditions existed [28].

The kinetic constants were determined at pH 6.8 from Lineweaver-Burk plots of initial velocity data (e.g. Fig. 2). The concentrations of C 0 2 and of HCO; in the assay were calculated from the amount of HCO; added by using the following three equa- tions: (a) HCO; added = HCO; in solution + C02 in solution + C02 in gas phase; (b) log ([HCO;] in solution/[CO,] in solution) = pH-pK; (c) C02 in solution/C02 in gas phase = (volume of liquid phase/ volume of gas phase)xcr; volume of gas phase = 3.8 mi; volume of liquid phase = 1 ml; pK = 6.3 [21]; a = 0.638 at 35 “C [32]. The calculation is based on the assumption that at pH 6.8 H2C03 and Cog- can be neglected and that at 35 “C and under continuous rapid shaking equilibrium was attained between the C02 in the gas phase, the C 0 2 in solution and the HCO, in solution. This assumption is valid as shown by the finding that at 35 “C the rate of ‘C02’ reduction to formate with reduced ferredoxin and the rate of isotopic exchange between ‘C02’ and formate were constant even when very low amounts of HCO; were added.

For formate oxidation to ‘C02’, benzyl viologen was routinely used as electron acceptor. The reduction of benzyl viologen was followed photometrically. The assays were carried out at 35 “C under strictly anaerobic conditions in 1-ml cuvettes closed with a rubber stopper. 1-ml assay mixtures were used leaving a gas phase of 0.35 ml. Anaerobic conditions were obtained by repeatedly evacuating and refilling the cuvettes with argon. The reaction was started by injection of enzyme solution with a Hamilton syringe. Benzyl viologen reduction was measured at 578 nm (c5,* = 8.7 mM-’ cm-’) using a Zeiss photometer PM4 in connection with a Metrawatt recorder Servo- gor RE 511.

Detailed assay conditions are given in the legends to the table and the figures.

Determination of [‘4C]Formate

The reactions were stopped by the addition of trichloroacetic acid as described above. In order to

remove residual 14C02 the test tubes were repeatedry evacuated and refilled with unlabeled COz. Aliquots of the solution containing the [14C]formate were counted in 15 ml Bray scintillator [29] using a Packard liquid scintillation spectrometer 2425. In several experiments [14C]formate was determined after separa- tion by isoionic-exchange chromatography [30]. With this technique it was secured that in the acid solution no I4C-labeled compound other than formate was present in significant amounts.

RESULTS

C02 reductase from C.pasteurianum is a very labile enzyme which is rapidly inactivated by only trace amounts of molecular oxygen. An efficient purification procedure for the enzyme has, until now, not been worked out. The active species of ‘C02’, i.e. C 0 2 or HCO; (H2C03) utilized by the C02 reductase could, however, be determined using the unpurified enzyme for two reasons: (a) the partially purified extracts were found to contain only very low amounts of carbonic anhydrase activity and (b) the C 0 2 reductase activity of the partially purified extracts was high enough to allow rate determinations even at temperatures as low as 0 “C.

Reactions Catalyzed by C 0 2 Reductase

The partially purified extracts of C. pasteurianum were found to catalyze the following reactions: (a) ‘C02’ reduction to formate with reduced ferredoxin, (b) isotopic exchange between ‘C02’ and formate in the absence of ferredoxin, and (c) formate oxidation to ‘C02’ with oxidized ferredoxin or benzyl viologen (Table 1). These reactions appear to be catalyzed by one enzyme. This is upheld by the finding that the three reactions are reversibly inhibited by low concentrations of azide (Ki = 4 pM) and that the three activities mediating the reactions are irreversibly inactivated by low concentrations of cyanide (10 pM) [31]. Therefore the active species of ‘C02’ utilized or formed in the three reactions should be the same.

The Active Species of ‘C02’ Utilized

The method of determining ‘C02’ species described by Cooper et al. [18] can be applied only to ‘C02’- consuming reactions and not to ‘C02’-forming reactions. The C 0 2 reductase from C. pasteurianum was shown to catalyze both a ‘C02’ reduction to formate with reduced ferredoxin and an isotopic exchange between ‘C02’ and formate in the absence of ferredoxin. Thus two reactions were available for the


114 The Active Species of TO,' Litilized by CO, Reductase

Table 1, Reactions cu tu l j i r r l by C'O, reductuse,from C. pasteurianum Assay for C 0 2 reduction: 100 mM imidazole acetate pW 6.8, .SO m M mercaptc!ethanol. 1 -- 10 pmol potassium ['4C]bicarbonate (220 dis. min- ' nniol~ '). 0.5 - 10 pM ferredoxin, partially purified extract containing COz reductase and ferredoxin hydrogenase (0.5-3 mg protein), H,O to 1 ml; gas phase, hydrogen; temperature, 35 ' C , reaction timc. 1 -- 5 min; determination of CO, reduction by measuring the formation of ['4C]formate. Assay for isotopic exchange: I00 niM imidazole acetate pH 6.8, SO mM mercaptoethanol, 1 ~- 10 pmol potassium ['4C]bicarbonate (220 dis. min-' nmol 0.1 -20 mbl potassium formate, partially purified extract (0.5-3 mg protein). H,O to 1 ml; gas phase, argon; temperature 35 "C; reaction time. 1 - 5 min; determination of exchange by measuring the formation of ["CC]formate. Assay for formate oxidation: 100 m M imidazole acetate pH 6.8, 50 mM mercaptoethanol, 0.5- 20 mM potassium formate, 0.1 -2 mM benzyl viologen. partially purified extract (0.25 mg protein), H,O to 1 ml; gas phase, argon: tcmperature 15 C; determination of formate oxidation by measuring the reduction of benzyl viologen photometrically

C'02 rl,dul.tioir CO, I' HCO; Ferredoxin

mUimg protein mM

47 0.3 1.7 0.003

I S C J ~ O ~ J ic ( 7 . t c,llcltl yc CO, a

48

HCO, I, Formate

0.5 2.3 0.5

Folmute 0uidor 1011

Formatc 2 Ferredoxin 45 Benzql viologeii 200 <0.01

-? Assuming C O , t o be the substrate. Assuming HCO, to be the substrate.

determination of the active species of 'CO,' utilized by CO, reductase from C. pasteuriunum.

The incorporation of I4C from '14C02' into formate was measured at 0 "C in the absence and the presence of carbonic anhydrase. The experimental results are shown in Fig. 1 . There was a high rate of I4C incorporated into formate in both the reduction reaction (Fig. 1 A) and the exchange reaction (Fig. 1 B) when C02 was the initially labeled species (curves designated 14C02) and a very low rate of incorporation when HCO, was the initially labeled species (curves designated H14CO;). In the presence of carbonic anhydrase the high initial rate of 14C incorporation observed when C02 was the species labeled initially was abolished and identical rates of I4C incorporation into formate were observed regardless of the species labeled initially (curves designated C. A,). These results are consistant only with C 0 2 as the active species utilized by CO, reductase.

t C . A . /O I /

J H'4C0G A

v- I I

0 5 10 15 20 Time (min)

Time (min)

Fig. 1. Radiocfiemic,ul U A , ~ U , J 14 CO, rrductri.w octiviries 11h1w eitllcJr HCO, p l u ~ l4CO, (a) (11' H'"CO, pi1r.r ( 0 , ioi 1wI.c t ~ i f c k d initially. (A) TO,' reduction to formate uith reduced fkrredoxin. Assay: 10 mM imidazolc acetate pH 7.2. 5 inhl dithiocrythritol, 10 pM ferredoxin, partially purified extract oi c'. /mtruruniuni containing CO, reductase and ferredoxin hydrogcnase (7.5 mg protein), 3 mM KHCO,, 3 mM CO, (formed b) equal quantities of KHC03 and acetic acid, the indicated radioactive species having a specific radioactivity of 4.4 dis. mill-' pniol-'1, H 2 0 to 3 ml: gas phase, hydrogen: temperature. 0 'C : 3 mg carbonic anhydrase (C.A.) was added where indicated; determination of 'C02' reduction by measuring the forination of ['4C]formatt., (B) Isotopic exchange between 'CO,' and formate in the absence of ferredoxin. Assay conditions as described above except 7hat 10 mM potassium formate was added instead of ferredoxin

In the presence of carbonic anhydrase the rate of CO, reduction to formate was alivays higher than the rate of isotopic exchange (Fig. 1 A and 1 B respec- tively). This difference cannot satisfactorily be explained.

Lur. .1. Biochem. 5.5 (1975)

R. K. Thauer, B. Kaufer, and G. Fuchs

a, c e

40

3 30

115

- -

l/[Formate] (mM-')

Fig. 2. Kinetics of isotopic exchange between CO, and formate in the absence offerredoxin at different fixed levels offormate ( A ) or CO, ( B ) . Assay: 100 mM imidazole acetate pH 6.8, 2 mM dithioerythritol, 1 - 10 pmol potassium ['4C]bicarbonate (220 dis. min-' nmol-') as indicated, 0.05 -20 mM potassium formate as indicated, partially purified extract (2.5 mg protein), H,O to 1 ml; gas phase, argon; temperature, 35 "C; reaction time 1 and 5 min; determination of exchange by measuring the formation of ['4C]formate; the concentration of CO, in the assay was calculated from the amount of HCO; added as described under Materials and Methods

p H Dependence of Kinetic Constants

The V and the dissociation constant K , of the enzyme . CO, complex were determined by measuring the rate of isotopic exchange between CO, and formate in the absence of ferredoxin at different CO, and formate concentrations. The data were plotted accord- ing to Lineweaver-Burk. At saturating concentrations of formate the intercept on the abscissa is equal to

PH Fig. 3. pH-dependence ofthe rate of isotopic exchange. (0) Morpho- linoethanesulfonic acid (100 mM) plus KOH ; (0) morpholinopropanesulfonic acid (100mM) plus KOH. The V values were determined as described in Fig. 2

3 t

2

I

I E - r"

1

0

-

-

I I I

5 6 7 PH

Fig. 4. pH-dependence of the dissociation constant (K,) of the enzyme . CO, complex. (0) Morpholinoethanesulfonic acid (100 mM) plus KOH; (0) morpholinopropanesulfonic acid (100 mM) plus KOH. The K, values were determined as described in Fig. 2

l / K , and the intercept on the ordinate is equal to l / V [28] (Fig. 2A and B).

The pH optimum was found to be broad with a maximum near 6.5 (Fig. 3). This is of interest as the intracellular pH in growing cultures of C. pasteuriunum has been shown to be between 6 and 7 [33] and as at this pH considerable amounts of CO, are always present.


116 The Active Species of ‘COz’ Utilized by C 0 2 Reductase

The dissociation constant of the enzyme . CO, complex increased with decreasing pH (Fig. 4). Experi- ments were performed down to a pH of 5.3. Measure- ments made below a pH of 5.5 could, however, not be evaluated as at pH values below 5.5 the enzyme was rapidly inactivated.

DISCUSSION

The present results indicate that CO, rather than HCO; (or H2C03) is the active species of ‘CO,’ utilized by CO, reductase from C. pasteurianum. Cooper et a/. [18] have, however, pointed out that it is conceivable that the method employed is not a reflection of the mechanism per se, but a reflection of the rate of binding at the active site. For example the charged HCO; might be hindered from approach- ing the active site and CO, might more readily approach the site. At the active site CO, might react with H 2 0 to form HCO; and in this form undergo the actual chemical reaction. The finding, however, that charged anions such as azide, cyanate and thiocyanate are effective inhibitors of CO, reductase competitive to CO, [31] makes such an interpretation unlikely.

Cooper et al. [18] have published theoretical curves for enzymes which utilize COz and for those which utilize HCO; as substrate. The theoretical curves derived for the situation when CO, is the species utilized are very similar to the experimental curves shown in Fig. 1. Essentially only the time scales of the figures are different. This difference can, however, easily be explained by the different temperatures used in the calculations and in the experiments. The theoretical curves have been calculated assuming a temperature of 25 “C. The curves in Fig. 1 , however, were measured at 0 “C. At 0 “C the time to attain equilibrium of the reaction H 2 0 + CO, + H2C03 is 15-fold larger than at 25 “C. This is due to the high Ql0 of approximately 3 of the hydration reaction [20].

The pH dependence of the dissociation constant of the enzyme . CO, complex (K,) observed suggests that a dissociable group with a pK of below or near 6 might be involved in CO, binding to the enzyme (Fig. 4). This could be the imidazole base of histidine or the o-carboxylate groups of aspartate or glutamate. Due to the failure to do complete kinetic experiments at pH values below 5.5 the participation of an acidic group in C02 binding is merely speculative.

The active species of ‘CO,’ utilized by other enzymes involved in ‘CO,’ fixation have recently been determined. CO, was found to be the substrate of phosphoenolpyruvate carboxykinase [18], of phosphoenolpyruvate carboxytransphosphorylase [ 181 and of ribulose bisphosphate carboxylase [34] while HCO;

appears to be the substrate of phosphoenolpyruvate carboxylase [35], of pyruvate carboxylase [18] and of propionyl-CoA carboxylase [36]. Thus neither CO, nor HCO; appears to be preferred as substrate in ‘COz’ fixation. The simultaneous requirement in one cell of both CO, and HCO; might explain why carbonic anhydrase is found almost ubiquitously

This work was supported by a Grant from the Deutsche For- schungsgemeinschaft, Bonn-Bad Godesberg. We wish to thank Dr K. Jungermann (Freiburg) for stimulating discussions.

[20,21].

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R. K. Thauer, B. Kaufer, and G. Fuchs, Abteilung fur Biologie der Ruhr-Universitat Bochum, D-4630 Bochum-Querenburg, Postfach 2148, Federal Republic of Germany


the active species of ‘co2’ utilized by reduced ferredoxin: co2 oxidoreductase from clostridium...

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