Principles of Bioinorganic ChemistryLecture Date Lecture Topic Reading Problems

1 9/4 (Th) Intro; Choice, Uptake, Assembly of Mn+ Ions Ch. 5 Ch. 12 9/ 9 (Tu) Metalloregulation of Gene Expression Ch. 6 Ch. 23 9/11 (Th) Metallochaperones; Metal Folding, X-linkingCh. 7 Ch. 34 9/16 (Tu) Zinc Fingers; Metal Folding; Cisplatin Ch. 8 Ch. 45 9/18 (Th) Cisplatin; Electron Transfer; Fundamentals Ch. 9 Ch. 56 9/23 (Tu) ET Units; Long-Distance Electron Transfer Ch. 9 Ch. 67 9/25 (Th) ET; Hydrolytic Enzymes, Zinc, Ni, Co Ch. 10 Ch. 78 10/ 7 (Tu) Model Complexes for Metallohydrolases Ch. 10 Ch. 89 10/ 9 (Th) Dioxygen Carriers: Hb, Mb, Hc, Hr Ch. 11 Ch. 910 10/10 (Fr)* O2 Carriers/Activation, Hydroxylation: MMO,

P-450, R2Ch. 11 Ch. 10

11 10/14 (Tu) Model Chemistry for O2 Carriers/Activators Ch. 12 Ch. 1112 10/16 (Th) Complex Systems: cyt. oxidase; nitrogenase Ch. 12 Ch. 1213 10/21 (Tu) Metalloneurochemistry/Medicinal Inorg. Chem.14 10/23 (Th) Term Examination

*Makeup class, 8:30 – 10 AM; room 2-135

You should have your paper topic approved by Prof. Lippard this week, if you have not done so already (by 10/12 please). The oral presentations will be held in research conference style at MIT's Endicott House estate

in Dedham, MA, on Saturday, October 18. WEB SITE: web.mit.edu/5.062/www/

Structural and Spin State Changes upon Binding of Dioxygen to an Iron Porphyrin

Center

High-spinferrous

Low-spinferric

Deoxy Hb (T state) Oxy Hb (R state). Hb binds 4 O2 molecules. When 2 are bound, T switches to R and makes the next ones easier to bind.

Model Chemistry for Oxy Hb and Oxy Mb

The problem:FeIIP + O2 FeIIIP–O2

- PFeIII–O O–FeIIIP

..

..2PFeIV=O: PFeIII–O–FeIIIP

.. FeIIP

-oxo, “dimer”

The solutions:Attach the porphyrin to a solid support to

avoid the bimolecular reaction; or, use low T, non-aqueous solvents, and py or 1-MeIm complexes, but stability is lost at - 45 °C or above. The best solution was the construction of a sterically hindered cavity for dioxygen binding to avoid the intemolecular chemistry leading to the thermodynamic sink of the system, the (-oxo)diiron(III) species.

FeIIP

ferryl

Synthetic Models for OxyHb and OxyMb

(Collman) (Baldwin)

The Cytochrome P-450 Reaction Cycle

When an axial site is available on the iron porphyrin, dioxygen can bind and/or be activated there. With proton-mediated reductive activation of the O2 molecule, a peroxo intermediate forms that converts to an FeIV=O species, the ferryl ion.

The ferryl can oxidize hydrocarbons to alcohols, epoxidize olefins, oxidize amines to amine oxides and do related chemistry.

P-450’s are liver enzymes necessaryfor metabolism and used to convertpro-drugs and pro-carcinogens totheir active forms.

Protoctechuate 3,4-Dioxygenase

+H2O

+

O2

- H2O+

O

OH

-OOC

OH

OH-OOC

O

OH

HO FeII I

O

N

O

His

NH

O

N

NH

HO

FeII IHis

-OOCO

O

OO

FeII I

O

HO

-OOCO

His

ON

NH

O

N

NH

O

FeII IHis

HOO

O

O

-OOC

O

OFeII-OOC

Notes: dioxygenase vs. monooxygenase; iron oxidation state does not change; iron acts as a Lewis acid; semiradical character of the catecholate ligand activates it for directattack by the dioxygen molecule.

Hemerythrins - Diiron Dioxygen Carriers

Properties:Mono- (myo Hr) and multi- (Hr) subunit proteins.Found in marine invertebrates.

Easily isolated protein; crystallizes after one step!!

Deoxy Hr, colorless, diiron(II)Oxy Hr, red, diiron(III) peroxo O–O, 844 cm-1 in the terminally bound peroxide region. Fe–O–Fe, 486 cm-1, resonance enhanced symmetric

stretch. The asymmetric stretch occurs at 757 cm-1.

Mixed-valent, semimet Hr, Fe(II)Fe(III): inactive.

Structure of AzidomethemerythrinContains a (-oxo)diiron(III) core. Met, artificially oxidized. An inactive form of the protein. The azido anion occupies the place of the hydroperoxo anion in oxyHr.

The structure was encountered for the first time when the protein crystallographers found it in azidometmyoHr. Myo, single subunit.

The electronic spectrum is characteristic and a consequence of antiferromagnetic spin exchange between the two high-spin Fe(III) centers.

FeIIIO

FeIII

OO

Glu

OO

Asp

N(His)

O N(His)(His)N

(His)N

(His)N

FeII

HO

FeII

OO

Glu

OO

Asp

N(His)

N(His)(His)N

(His)N

(His)N

OH

O2

DeoxyHrDiferrous

OxyHrDiferric

Hydrophobic Residues

Chemistry at the Active Site of Hemerythrin (Hr)

Note proton-coupled electron transferEvidence for proton transfer comes from resonance Raman work

Early Structural Models for Methemerythrin

R

O ON

FeO

Fe

O O

N

N

NN N

R

Wieghardt, N3 = Me3TACNArmstrong, Lippard, N3 = HB(Pz)3-

(- ) ( ) Carboxylato diiron III Complexes

R

O ON

FeO

Fe

O O

N

N

NN N

R

2+

These and related complexes have no site for binding of azide or dioxygen related species such as hydroperoxide.

The syntheses exemplify spontaneous self-assembly.

The challenges are to make a site available, allow redox chemistry to occur, and avoid polymerization to rust or molecular ferric wheels and related complexes.

O

O

OH

O O

HO

PhPh

PhPh

O

OH HO PhPh

PhPh

O

HHPhPh Ph

Ph

Synthesis of H2Ph4DBA

H2Ph4DBA40% Overall Yield

i) n-BuLi/TMEDA/hexaneii) Ph2COiii) HCl(aq)

i) Et3SiH/CH2Cl2ii) BF3·Et2Oiii) Na2CO3(aq)

i) n-BuLi/THFii) CO2(g)iii) HCl(aq)

Mizoguchi

None does the chemistry of the protein!

0

2000

4000

6000

8000

300 400 500 600 700 800 ( )Wavelength nm

- UV Visible Absorption Changes

Diferrous

HYDROPEROXIDE

+ O2

λmax

≈ 470 nm

εmax

≈ 2500 M-1 cm-1

780800820840860

(Resonance Ramanλex = 514.5 )nm

(Raman Shift cm-1)

(16 –O 16 )O

843 cm-1

(18 –O 18 )O

797 cm-1

Fe

HO

Fe

O OO O

N

OTfN

N

N

FeO

Fe

O OO O

N

NN

N

NO

NMe

HO

Spectral Properties of an OxyHr Model

+O2

3 equiv N -MeIm

CH 2Cl2, -78 °C

Red-OrangeSolution

OxyHr: (16O—16O) = 844 cm-1 λmax, 500 nm

(18O—18O) = 798 cm-1

Properties of Oxy Hr, Deoxy Hr, and Models

Structure and Chemistry of Class I Ribonucleotide Reductase R2 Protein

Reaction of the reduced diiron(II) form of the R2 protein with dioxygen affords a high valent, Fe(III)Fe(IV) intermediate designated as X. Intermediate X is kinetically competent to oxidize the tyrosyl residue to afford a tyrosyl radical. This radical in turn transfers electrons to the R1 subunit of the enzyme where a Cys-S–S-Cys cation radical forms. This radical in turn initiates chemistry to convert ribo- to deoxyribonucleotides.

Hemocyanins - Dicopper Dioxygen Carriers

Properties:Multi-subunit proteins, ranging in size up to 460 kDa.Found in spiny lobsters, crayfish, and arachnids.

Deoxy Hc, colorless, dicopper(I)Oxy Hc, blue, dicopper(II) peroxide O–O, 745-750 cm-1 in the peroxide region, but low.

Unusual structure, first established by model chemistry: O

Cu Cu O

Schematic Views of Deoxy and Oxy Hc

Note, Type III copper

Structure of Deoxyhemocyanin

The two Cu atoms are held by six terminal histidine residues, the Cu Cu distance being 3.7 Å. There is no obvious bridging ligand.

...

Monooxygenase Activity in Synthetic Cu2 Models

The dinuclear complex mediates insertion into the C–H bond. The chemistry mimics that of tyrosinase.

Important Relationships

Reversible O2 binding

•Iron porphyrin, Hb/Mb Iron porphyrin, P-450

•Dicopper center, Hc Dicopper center, tyrosinase

•Diiron center, Hr Diiron center, R2, MMO

O2 Activation

WHAT CONTROLS THE FUNCTION??

Principles Illustrated by these Cases

Substrate binding and redox changes occur:

•In all three cases, O2 binding is accompanied by electron transfer from one or two metal ions to dioxygen.Coupled proton-electron transfer steps set the potentials:•In oxyHr a proton transfers from the bridging hydroxide to the peroxo ligand; this step appears to block further conversion to high-valent iron oxidase center(s).Metal center used to create or destroy radical species:•Occurs in ribonucleotide reductase R2 protein. Catechol dioxygenase - Fe(III) coordination favors semiquinone form of a bound ligand without redox reaction occurring.Changes in metal coordination sphere facilitate allostery:•Explains the cooperativity of O2 binding in Hb.

CH4 CH3OH

O2 H2O

H2CO

HCOOH

CO2

NADH + H+

Methane monooxygenase (MMO)

Formal-dehyde

dehydro-genase

Formate dehydro-genase

Oxidation of Methane in Methanotrophs

ribulose monophosphate pathway

Carbon assimilation

Type II

serine pathway

Type I, Type X

Type I - Methylomonas methanica ♦ ( )Particulate MMO Cu ♦ rod shaped ♦ 30 °growth at C ♦ bundled membranes

Type X- Methylococcus capsulatus ( )Bath ♦Particulate and soluble MMO depending on growth conditions ♦spherical ♦growth at 45 °C ♦bundled membranes

Type II - Methylosinus trichosporium OB3b ♦ Particulate and soluble MMO ♦ rod shaped ♦ growth at 30 °C ♦ paired membranes

Methanol dehydro- genase

Plants recruit oil-detoxifying microbes, as discovered by scientists analyzing the recovery of the environment in the Persian Gulf region following the 1991 Gulf War.

" In the root zone was a rich reservoir of well-known oil eating microbes...one family of which (Arthrobacter) accounted for fully 95 percent..."

Science News, 148, 84 (August 5, 1995)

Methanotrophs are Used in Bioremediation of the Environment

Prince William Sound, Alaska:

After the Exxon Valdez oil spill, fertilizers were spread on the beaches and natural methanotrophs restored their pristine beauty.

The Mineral Springs in Bath, England,Source of Methylococcus capsulatus (Bath)

The Restutive Contents of the WATER’s Concoctive Power: Solution of gaffes, chaos of Salts and mineral effluvia of subterranean expiration. It cleanses the body from all blotches, scurvicial itchings and BREAKING OUTS WHATSOEVER!

B

Hox

Coupling Protein

Hydroxylase

Reductase

Hred

N A D H + H+

NAD+

O2

CH3OH + H2O

CH4

FAD

Fe FeS

S

B

Hactivated

Research Objectives for the sMMO System

Determine structures of component proteinsTrack electron transferthrough MMOR to thediiron center in MMOHElucidate the hydrocarbon

oxidation mechanism

Characterize intermediates in thedioxygen activation pathway Investigate roles of protein B

In addition, develop synthetic models to interrogate aspectsof the MMOH active site chemistry

Structures of the sMMO Components