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Principles of Bioinorganic Chemistry - 2004. Note: The course seminar presentations will be held on Sunday, October 31, 2004 in the Bush Room. Please remember that daylight savings time ends that day. Dioxygen Carriers: Hb, Mb, Hc, Hr Examples of Atom- and Group-Transfer Chemistry. PRINCIPLES:.

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Principles of Bioinorganic Chemistry - 2004

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    1. Principles of Bioinorganic Chemistry - 2004 Note: The course seminar presentations will be held on Sunday, October 31, 2004 in the Bush Room. Please remember that daylight savings time ends that day.

    2. Dioxygen Carriers: Hb, Mb, Hc, Hr Examples of Atom- and Group-Transfer Chemistry PRINCIPLES: • Both substrate binding and redox changes occur • Coupled proton-electron transfer steps set the redox potentials • Closely positioned redox/acid-base units work in concert • Interactions with substrates/other proteins gate electron transfer • Two-electron transfer strategies include 2 metals, M-porphyrins • Metal centers used to create or destroy radical species • Changes in metal coordination spheres can facilitate allostery • Bioinorganic chemistry of dioxygen paramount example ILLUSTRATIONS: • O2 Binding and Transport: hemoglobin (Hb), myoglobin (Mb), hemocyanin (Hc), and hemerythrin (Hr) • O2 Activation: cytochrome P-450, tyrosinase, methane monooxygenase; dioxygenases

    3. Properties of Protein Dioxygen Carriers

    4. Structure of Myoglobin proximal side Fe held into the protein solely by His imH ring. Deoxy structure has Fe out of plane of ring by 0.42 Å toward the proximal side of the porphyrin. Upon O2 binding, Fe moves into ring plane. distal side

    5. Vibrational Spectroscopic Evidence that OxyHb and OxyMb are Formally FeIII–O2- Species From resonance Raman spectroscopy the O–O stretch in oxyMb is measured to be ~ 1105 cm-1. The protein is also diamagnetic (d5, Fe(III) and O2- couple).

    6. Structural and Spin State Changes upon Binding of Dioxygen to an Iron Porphyrin Center 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. High-spin ferrous Low-spin ferric

    7. Model Chemistry for Oxy Hb and Oxy Mb The problem: FeIIP + O2 FeIIIP–O2- PFeIII–O O–FeIIIP .. FeIIP .. .. FeIIP 2PFeIV=O: PFeIII–O–FeIIIP m-oxo, “dimer” ferryl 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 (m-oxo)diiron(III) species.

    8. Synthetic Models for OxyHb and OxyMb (Collman) (Baldwin)

    9. Structure of Azidomethemerythrin Contains a (m-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.

    10. 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 nO–O, 844 cm-1 in the terminally bound peroxide region. nFe–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.

    11. Note proton-coupled electron transfer Evidence for proton transfer comes from resonance Raman work

    12. Early Structural Models for Methemerythrin 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.

    13. Early Structural Models for Deoxyhemerythrin None does the chemistry of the protein!

    14. Properties of Oxy Hr, Deoxy Hr, and Models

    15. Dioxygen Binding Chemistry of the Hr Model Complex Product matches protein Mössbauer, resonance Raman, UV-vis spectra

    16. 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 nO–O, 745-750 cm-1 in the peroxide region, but low. Unusual structure, first established by model chemistry: O Cu Cu O

    17. 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. ...

    18. Schematic Views of Deoxy and Oxy Hc Note, Type III copper

    19. Model Chemistry for Deoxy and Oxy Hc Karlin model Kitajima model

    20. Important Relationships Reversible O2 binding O2 Activation • Iron porphyrin, Hb/Mb Iron porphyrin, P-450 • Dicopper center, Hc Dicopper center, tyrosinase • Diiron center, Hr Diiron center, R2, MMO WHAT CONTROLS THE FUNCTION??

    21. 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 necessary for metabolism and used to convert pro-drugs and pro-carcinogens to their active forms.

    22. 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. Changes in metal coordination sphere facilitate allostery: Explains the cooperativity of O2 binding in Hb.

    23. The Mineral Springs in Bath, England, Source of Methylococcus capsulatus (Bath) The Restitutive 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, scurvical itchings and BREAKING OUTS WHATSOEVER!

    24. Methanotrophs are Used in Bioremediation Prince William Sound, Alaska: After the Exxon Valdez oil spill, fertilizers were spread on the beaches and natural methanotrophs restored their pristine beauty. 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)

    25. Evolutionary Relationships Between Multicomponent Monooxygenases Common Ancestor Phenol Hydroxylases Amo Alkene Monooxygenase Dimethyl Sulfde Hydroxylase Phenol Hydroxylase Soluble Methane Monooxygenases Four Component Alkene/ Aromatic Monooxygenases Methane Monooxygenase Butane Monooxygenase Toluene Monooxygenases Phenol Hydroxylases Isoprene Monooxygenases

    26. Properties of Hydrocarbon Monooxygenases Containing Carboxylate-Bridged Diiron Centers Leahy, Batchelor, Morcomb, FEMS Microbiol. Rev.2003, 770, 1-31.

    27. Properties of Methanotrophs pMMO, Cu sMMO, Fe • 5-50 Tg CH4/year consumed by soil methanotrophs (1-10% of • atmospheric CH4), converting this greenhouse gas to biomass. • 104 kcal/mol BDE for methane makes it a challenge to activate. • Controlled oxidation to methanol at moderate temperatures in neutral aqueous solution is a remarkable chemical feat. • 500 billion barrels crude oil equivalent in recoverable but remote natural gas deposits might be made available.

    28. Carboxylate-Bridged Diiron Proteins Global Research Goals • What tunes the properties of the diiron centers? • What are the electron transport pathways? • What factors control dioxygen reactivity? • How is substrate specificity achieved? Objectives for the sMMO and Model Studies • Determine structures of all components and complexes • Understand hydroxylation and epoxidation reactions • Synthesize and characterize structural/spectroscopic models • Achieve selective oxidation and catalysis

    29. Soluble Methane Monooxygenase (sMMO) Component Structures & Reaction Cycle

    30. sMMO is a Multicomponent Enzyme • Reductase • MMOR • Uses FAD and [2Fe-2S] for electron transfer from NADH to MMOH • Hydroxylase • MMOH • a2 b2 g2 • Dinuclear iron active site in each a subunit • Hydroxylation chemistry • Regulatory Protein • MMOB • required for full activity

    31. MMOH Dinuclear Iron Active Site Glu209 Glu243 Glu114 His246 His147 Glu144 Hox, (FeIII)2 Hred, (FeII)2 Both Hox and Hred are charge neutral; X-ray structures by Rosenzweig, Whittington, et al., 1993-present

    32. The Catalytic Cycle of sMMO H2O NAD+ O2 NADH MMOR B MMOHred MMOHsuperoxo MMOHox B B H+ MMOHperoxo Mössbauer (d 0.66 mm s-1) UV-vis, 725, 410 nm MMOHQ Mössbauer (d 0.17 mm s-1) UV-vis, 420 nm; EXAFS CH3OH CH4 RH RH(O) B

    33. B Reactions of CH3X Substrates with Q H+ MMOHQ Mössbauer (d 0.17 mm s-1) UV-vis, 420 nm; EXAFS CH3OH CH4

    34. Reaction of Q with Methane by Double-Mixing SF kH/kD = 26 CH4 T = 20 ºC [H]red = 16.8 µM [CH4] = 0.50 mM kobs = 14.1(1) s-1 CD4

    35. Mechanism for Methanol Formation E = 0.0 kcal/mol Q Methane Gherman, Dunietz, Whittington, Lippard & Friesner, J. Am. Chem. Soc. 2001, 123, 3836. Baik, Gherman, Friesner & Lippard, J. Am. Chem. Soc., 2002, 124, 14608.

    36. First Electron Transfer for Methanol Formation 17.9 kcal/mol • First electron transfer occurs here and determines the barrier height; one Fe reduced to Fe(III) as O–H bond forms.

    37. Mechanism for Methanol Formation This transition state is 1.3 kcal/mol uphill from the bound radical intermediate, affording a rate constant in accord with most radical clock substrate probe studies.

    38. Electronic Details of Second Electron Transfer Acceptor Orbital Donor Orbital Bound Methyl Radical (b-Spin) Fe1 d-(x2-y2) (b-Spin-LUMO) ‘Mediator’ Orbital Oxo p(z) (doubly occupied) Baik, Gherman, Friesner & Lippard, J. Am. Chem. Soc.2002, 124, 14608.

    39. Electronic Details of Second Electron Transfer •H–O rotation promotes intramolecular b-electron transfer from the oxo lone pair orbital to the metal-based LUMO. •The remaining radicaloid a-electron on the bridging oxo group has the correct spin to recombine with the b-electron on the substrate to form a s-bond.

    40. Overall Energetics and Methanol Release 1.3 -69.7 E in kcal/mol

    41. Reactions of Q with Substrates Reveal Complexities C2H6 CH4 C2D6 CD4 Puzzles: This result indicates that, for ethane, the rate-determining step in not C–H bond activation. Yet kobs is the same! Answers: For CH4, H atom abstraction is rate determining; for C2H6, binding is rate determining. The bond in C2H6 is weaker, lowering of the C–H bond activation energy by ~5.6 kcal/mol, from both experiment and theory.

    42. kobs vs Nitromethane Concentration for Q Decay Solid circles, CH3NO2 Open circles CD3NO2 pH = 7, 20 C ; KIE, 8.1 Direct evidence for bound substrate in a Q reaction is facilitated by the high solubility of nitromethane. Ambundo, E. A.; Friesner, R. A.; Lippard, S. J. J. Am. Chem. Soc.2002, 124, 8770-8771.

    43. Single Turnover of Qwith Nitromethane-d3 at 25°C by Stopped-Flow Infrared Spectroscopy Loss of nitromethane-d3 monitored by stopped-flow IR spectroscopy at 1548 cm-1; kobs 0.39 s-1 Loss of Q monitored by stopped-flow spectrophotometry at 420 nm; kobs 0.39 s-1 First direct monitoring of the hydroxylation of a methane-derived substrate in the sMMOH reaction pathway Muthusamy, M.; Ambundo, E. A.; George, S. J.; Lippard, S. J.; and Thorneley, R. N. F. J. Am. Chem. Soc.2003, 125, 11150-11151.

    44. KIE for Reactions of Q with CH3X Substratesa CLASS I SUBSTRATES H atom abstraction rate-determining: CH4, CH3CN, CH3NO2 CLASS II SUBSTRATES Binding rate-determining: C2H6, CH3OH Ambundo, E. A.; Friesner, R. A.; Lippard, S. J. J. Am. Chem. Soc.2002, 124, 8770-8771.

    45. Gherman, B. F., Lippard, S. J., Friesner, R. A., submitted, 2004

    46. RH RH(O) Reactions of Substrates with Hperoxo MMOHperoxo Mössbauer (d 0.66 mm s-1) UV-vis, 725, 410 nm

    47. Preliminary Evidence for Hperoxo Reacting with Substrates filled circles - propylene open circles – methane Hperoxo appears to react with propylene Low solubility of substrates limits experiment Could propylene accelerate the conversion of Hperoxo to Q? Valentine, A. M.; Stahl, S. S.; Lippard, S. J. J. Am. Chem. Soc.1999, 121, 3876-3887.

    48. l 705 or 720 nm l 420 nm Conditions: T = 20 ºC, [H]red = 51.5 mM, [B] = 103 mM Ether concentration in excess and variable, 3 - 70 mM Hperoxo and Q Reactions with Ethyl Vinyl Ether The product of propylene reaction with Hperoxo is propylene oxide