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: O 2 Binding and Transport: hemoglobin (Hb), myoglobin (Mb), hemocyanin (Hc), and hemerythrin (Hr) O 2 Activation: cytochrome P-450, tyrosinase, methane monooxygenase; dioxygenases

3. Properties of Protein Dioxygen Carriers

4. Structure of Myoglobin 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 O 2 binding, Fe moves into ring plane. distal side proximal side

5. Vibrational Spectroscopic Evidence that OxyHb and OxyMb are Formally Fe III –O 2 - Species From resonance Raman spectroscopy the O–O stretch in oxyMb is measured to be ~ 1105 cm -1. The protein is also diamagnetic (d 5, Fe(III) and O 2 - couple).

6. Structural and Spin State Changes upon Binding of Dioxygen to an Iron Porphyrin Center High-spin ferrous Low-spin ferric Deoxy Hb (T state) Oxy Hb (R state). Hb binds 4 O 2 molecules. When 2 are bound, T switches to R and makes the next ones easier to bind.

7. Model Chemistry for Oxy Hb and Oxy Mb The problem: Fe II P + O 2 Fe III P–O 2 - PFe III –O O–Fe III P.. 2PFe IV =O: PFe III –O–Fe III P.. Fe II P  -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. Fe II P ferryl

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

9. Structure of Azidomethemerythrin Contains 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.

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 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.

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

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

17. 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 O

18. 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....

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

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

21. Important Relationships Reversible O 2 binding Iron porphyrin, Hb/Mb Iron porphyrin, P-450 Dicopper center, Hc Dicopper center, tyrosinase Diiron center, Hr Diiron center, R2, MMO O 2 Activation WHAT CONTROLS THE FUNCTION??

22. 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 O 2 molecule, a peroxo intermediate forms that converts to an Fe IV =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.

23. Principles Illustrated by these Cases Substrate binding and redox changes occur: In all three cases, O 2 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 O 2 binding in Hb.

24. 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!

25. 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 Prince William Sound, Alaska: After the Exxon Valdez oil spill, fertilizers were spread on the beaches and natural methanotrophs restored their pristine beauty.

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

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

28. Properties of Methanotrophs pMMO, Cu sMMO, Fe 5-50 Tg CH 4 /year consumed by soil methanotrophs (1-10% of atmospheric CH 4 ), 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.

29. Carboxylate-Bridged Diiron Proteins 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 Global Research Goals

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

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

32. H ox, (Fe III ) 2 Glu209 His246 His147 Glu144 Glu114 Glu243 MMOH Dinuclear Iron Active Site H red, (Fe II ) 2 Both H ox and H red are charge neutral; X-ray structures by Rosenzweig, Whittington, et al., 1993-present

33. NADH NAD + MMOR H2OH2O CH 4 CH 3 OH MMOH ox B H+H+ The Catalytic Cycle of sMMO B MMOH red MMOH superoxo O2O2 B MMOH peroxo Mössbauer (  0.66 mm s -1 ) UV-vis, 725, 410 nm RH MMOH Q Mössbauer (  0.17 mm s -1 ) UV-vis, 420 nm; EXAFS B RH(O)

34. Reactions of CH 3 X Substrates with Q MMOH Q Mössbauer (  0.17 mm s -1 ) UV-vis, 420 nm; EXAFS B CH 4 CH 3 OH H+H+

36. CH 4 CD 4 k H /k D = 26 T = 20 ºC [H] red = 16.8 µM [CH 4 ] = 0.50 mM k obs = 14.1(1) s -1 Reaction of Q with Methane by Double-Mixing SF

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

38. 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.

39. 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.

40. Electronic Details of Second Electron Transfer Donor Orbital Bound Methyl Radical (  -Spin) Acceptor Orbital Fe1 d-(x 2 -y 2 ) (  -Spin-LUMO) ‘Mediator’ Orbital Oxo p(z) (doubly occupied) Baik, Gherman, Friesner & Lippard, J. Am. Chem. Soc. 2002, 124, 14608.

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

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

43. Reactions of Q with Substrates Reveal Complexities CH 4 CD 4 C2D6C2D6 Puzzles: This result indicates that, for ethane, the rate-determining step in not C–H bond activation. Yet k obs is the same! Answers: For CH 4, H atom abstraction is rate determining; for C 2 H 6, binding is rate determining. The bond in C 2 H 6 is weaker, lowering of the C–H bond activation energy by ~5.6 kcal/mol, from both experiment and theory. C2H6C2H6

44. k obs vs Nitromethane Concentration for Q Decay 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. Solid circles, CH 3 NO 2 Open circles CD 3 NO 2 pH = 7, 20  C ; KIE, 8.1

45. Single Turnover of Q with Nitromethane-d 3 at 25°C by Stopped-Flow Infrared Spectroscopy Loss of Q monitored by stopped- flow spectrophotometry at 420 nm; k obs 0.39 s -1 Loss of nitromethane-d 3 monitored by stopped-flow IR spectroscopy at 1548 cm -1 ; k obs 0.39 s -1 Muthusamy, M.; Ambundo, E. A.; George, S. J.; Lippard, S. J.; and Thorneley, R. N. F. J. Am. Chem. Soc. 2003, 125, 11150-11151. First direct monitoring of the hydroxylation of a methane- derived substrate in the sMMOH reaction pathway

46. KIE for Reactions of Q with CH 3 X Substrates a CLASS I SUBSTRATES H atom abstraction rate-determining: CH 4, CH 3 CN, CH 3 NO 2 CLASS II SUBSTRATES Binding rate-determining: C 2 H 6, CH 3 OH Ambundo, E. A.; Friesner, R. A.; Lippard, S. J. J. Am. Chem. Soc. 2002, 124, 8770-8771.

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

48. Reactions of Substrates with H peroxo MMOH peroxo Mössbauer (  0.66 mm s -1 ) UV-vis, 725, 410 nm RH RH(O)

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

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

51. Results for H peroxo and Q with Ethyl Vinyl Ether Second Order Rate Constant k = 1500(100) M -1 s -1 Second Order Rate Constant k = 223(10) M -1 s -1 Rate constant for H peroxo is significantly greater than for Q. Diethyl ether reacts with Q reacts but H peroxo does not. Conclusion: H peroxo does react with substrates; others are under investigation. Beauvais, Bautista, and Lippard (2004), in preparation

52. Mechanisms for Epoxidation of Ethyl Vinyl Ether By H peroxo : By Q: H peroxo may react by 2-electron transfer and Q by single-electron transfer. In addition, there might be distinct binding and activation steps.

53. Substrate Access in MMOH and ToMOH B MMOH red MMOH superoxo O2O2 B CH 4 CH 3 OH MMOH ox B H+H+ B MMOH peroxo RH RH(O)

54. Toluene /o-Xylene Monooxygenase from Pseudomonas stutzeri OX1 Fe Rieske regulation ToMOB ToMOR Fe 2 S 2 ToMOH e-e- FAD Fe S S e-e- e-e- NADH NAD + + O 2    All components are expressed recombinantly in E. coli! ToMO has relaxed substrate specificity Substrates: toluene, xylenes, benzene, napthalene, phenol, cresols, ethylbenzene, styrene, halogenated ethylenes Substrates: toluene, xylenes, benzene, napthalene, phenol, cresols, ethylbenzene, styrene, halogenated ethylenes Fe Carfaro, V., et. al. Eur. J. Biochem. 2002, 296, 5689-5699.

55. M. H. Sazinsky, J. Bard, A. Di Donato, & S. J. Lippard, J. Biol. Chem. 2004, 279, 30600-30610. Crystallization of ToMOH Crystal Facts: Space group P3 1 21 Unit cell (Å) 182 x 182 x 67 Conditions: ± ToMOB ± NaN 3 (antimicrobial) Crystal Facts: Space group P3 1 21 Unit cell (Å) 182 x 182 x 67 Conditions: ± ToMOB ± NaN 3 (antimicrobial) 2F o -F c map from SAD Phasing 0.7 mm

56. Crystal Structures of ToMOH and MMOH The positions of the  subunit differs for the two hydroxylases. The folds of the  and  subunits are conserved; those of  are not. The canyon region in ToMOH is larger with a different aperture. Fewer    protomer contacts occur for ToMOH. The positions of the  subunit differs for the two hydroxylases. The folds of the  and  subunits are conserved; those of  are not. The canyon region in ToMOH is larger with a different aperture. Fewer    protomer contacts occur for ToMOH. MMOH ToMOH  -subunits

57. ToMOH ox + thioglycolate Active Site Structures of ToMOH ox and MMOH ox MMOH ox + acetate ToMOH has OH - and RCO 2 - bridges similar to those of MMOH and T4MOH. Spectroscopy of H ox T4MOHMMOHPH Absorption maxima (nm) Mössbauer , E Q (mm/s) 280 0.51, 0.93 (85%) 0.56, 1.55 (15%) 280 0.52, 1.16 0.51, 0.91 280, 350 0.54, 1.73 (85%) 0.48, 0.79 (15%) Pikus, J. D. et. al. Biochemistry. (1996), 35, 9106-9119. Cadieux, E., et al. Biochemistry. (2002), 41, 10680-10691. Fe-Fe distance 3.0Å

58. ToMOH has a Surface Accessible Channel  Channel is 35-40 Å long, 6-10 Å wide  Big enough for substrates and products  Diiron center has direct access to surface Fe Opening 1 Opening 2 Fork Openings

59.  ToMOH and MMOH use the same pathways for substrate entrance and product egress.  The channels and cavities may be a universal feature of all BMMs.  ToMOH and MMOH use the same pathways for substrate entrance and product egress.  The channels and cavities may be a universal feature of all BMMs. 4-Bromophenol in the ToMOH channel Universal Pathways for Small Molecule Access CH 2 Br 2 in MMOH cavities 2 and 3

60. Substrate Access at the Diiron Center F188 L110 Helix E Helix B Helix F Helix C Helix E Helix F Helix B Helix C I239 I110 F176 F196 F205 “The most prominent structural differences …is in an altered side chain conformation for Leu 110 at the active site cavity. We suggest that this residue serves as one component of a hydrophobic gate controlling access of substrates to and products from the active site.” -Rosenzweig, et al., Proteins 1997 29, 141-152 TomoHMMOH The ToMOH Fe 2 center is more accessible to solvent.

61. Electron/H + Transfer in MMOH and ToMOH NADH NAD + MMOR H2OH2O MMOH ox B B MMOH red

62. Testing the Role of an Absolutely Conserved Asparagine Helix E Helix B Helix E Helix B Helix C Helix F E240 N214 H147 BrEtOH T213 E243 Fe1 E114 - OH H ox + Bromoethanol H red Asn 214 is 100% conserved among all multicomponent monoxygenases. Cloning potential offers a chance to test function of N202 in ToMO. Could the role be to facilitate electron transfer? Proton transfer?

63. Steady-State Turnover of WT and N202A ToMOH Specific Activity (nmol/min/ mg) 19% N202A 25% N202A WTWT WTWT Single turnover results for ToMOH red + O 2 + ToMOD + phenol reveal no difference between wild type and N202A mutant protein activities. Working hypothesis: E. T. or H + pathway involves N202 in the ToMO system. (E. R. Cadieux and S. J. Lippard, Unpublished results (2004))