1. Principles of Bioinorganic Chemistry - 2003 The grade for this course will be determined by a term exam (35%), a written research paper with oral presentation (45%), problem sets (12%) and classroom participation (8%). The oral presentations will be held in research conference style at MIT's Endicott House estate in Dedham, MA, on Saturday, October 18. Please reserve the date for there are no excused absences. Papers will be due approximately one week earlier. WEB SITE: web.mit.edu/5.062/www/

2. Artificial Donor-Acceptor Pairs Cytochrome c; Fe---Ru, ~12 Å

3. Method for Studying ET of Ru-Modified Proteins Rate ~ 30 s -1, T-independent

4. Distance and Driving Force Dependencies of ET Rates

5. Driving Force Dependence Data are from ruthenium-modified cytochrome c derivatives (upper) and a series of covalently linked donor/acceptor compounds

6. Distance dependence from the T DA term  from the slope is 1.4 Å - 1. Get a 10-fold decrease in rate for every 1.7 Å increase in distance For comparison,  for ET in vacuum is 2.8 Å -1 and  for ET through covalent bonds is 0.7 Å -1 (thanks to Brian Crane for the plot)

7. 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!

9. NMR Structure of the Fd Domain of MMOR Mueller, Biochemistry, 41, 42-51 (2002)

11. Optical Spectra of MMOR and its Fd and FAD Domains MMOR oxidized Fd FAD Reduced forms

12. Redox States of the FAD Cofactor

13. Each trace is fit as a sum of exponentials giving rise to the reported rate constants.

15. Three major metallic units transfer electrons in bioinorganic chemistry: iron-sulfur clusters; blue copper including the dinuclear Cu A ; and cytochromes (iron porphyrins). Electrons can transfer over long distances in ~10-15 Å hops. The rate depends on driving force, distance, and orientation of the reacting partners. Pathways are important (  >  > H-bonds according to theoretical models). Electron transfer within and between proteins is optimized to take advantage of the molecular switching stations. Included are organic units such as flavins and inorganic units such as iron-sulfur clusters, both used in the MMOR protein. Summary - Points to Remember

16. Hydrolytic Enzymes, Zinc and other Metal Ions PRINCIPLES: M(OH) n+ centers supply OH - at pH 7 by lowering water pK a M n+ serves as general Lewis acid, activating substrates Rate acceleration occurs by internal attack within coord. sphere Protein side chains greatly assist assembly of transition state Carboxylate shifts can occur, especially at dimetallic centers Electrostatic interactions predominate Non-redox active metal ions often but not universally used Illustrating the Principles: Carboxypeptidase, carbonic anhydrase - delivering hydroxide Alcohol dehydrogenase: an oxidoreductase Dimetallic metallohydrolases: are two metals better than one?

17. Carboxypeptidase A: A Hydrolytic Zinc Enzyme Reaction catalyzed: R–CH–C(O)–NH–R’ NH 2 R’’ R–CH–CO 2 - + + NH 3 –R’ NH 2 R’’ Cleaves C-terminal peptide bonds; prefers aromatic residues. Active site contains a single catalytic zinc, essential for activity. The glutamate can undergo a carboxylate shift. Thermolysin has a similar active site; it is an endopeptidase.

18. Carboxypeptidase A structure with the inhibitor glycyl-L-tyrosine bound at the active site. Note hydrogen bonds to key residues in the active site that position the substrate moiety for bond scission.

19. Catalytic Mechanism for Carboxypeptidase A Summary of events: 1. Substrate binds; orients by the terminal carboxylate. 2. Deprotonate bound H 2 O. 3. Polarize scissile bond by Arg127. 4. Bound OH- attacks peptide C(O). 5. Form tetrahedral transition state. 6.Lose 2 peptide fragments and recycle the enzyme. Principles illustrated: 1. Zinc serves as template. 2.Metal supplies cleaving reagent, OH -, and organizes key groups. 3. Chemistry achieved at neutral pH! K cat ~ 100 s -1.

20. Carbonic Anhydrase, the First Known Zn Enzyme Reaction catalyzed: CO 2 + H 2 OH 2 CO 3 ~ 10 6 s -1

21. Note: Rate 10 -2 s -1 at pH 7; k f 10 6 s -1 in active site. Paradox: The reverse reaction is diffusion controlled, with k r ~ 10 11 M -1 s -1 Thus k f ≤ 10 4 s -1. So how can the turnover be 10 6 s -1 ? Answer: Facilitated diffusion of protons by buffer components bound to the enzyme. PZn(OH 2 ) 2+ PZn(OH) + + H + K eq = 10 -7 M = k f /k r Carbonic Anhydrase

22. Possible Carbonic Anhydrase Mechanism

23. Alcohol Dehydrogenase, an Oxidoreductase Reaction catalyzed: RCH 2 OH + NAD + RCHO + NADH + H + Enzyme contains two 40 kDa polypeptides, each with 2 Zn 2+ centers in separate domains. One zinc is structural, the other catalytic. Catalytic zinc is 20 Å from the surface, near the nicotinamide binding region. This center is not required for NAD + cofactor binding. Alcohol substate DO require zinc and bind directly to the metal center, displacing the coordinated water.

24. Schematic Diagram NAD + binding to the active site of LADH, with specific, well- positioned amino acid side chains holding it in place. Ethanol is shown bound to the zinc, displacing water. The system is set to undergo catalysis.

25. Note hydride transfers from  -C of alcohol to nicotinamide ring.

27. Dimetallics can move the value into the physiological range near pH 7

28. Advantages of Carboxylate-Bridged Dimetallic Centers in Chemistry and Biology

34. Alkaline Phosphatase; a Dizinc(II) Center Activates the Substrate 1. The substrate binds to the dizinc center; a nearby Arg also helps activate it. 2. A serine hydroxyl group attack the phosphoryl group, cleaving the ester. The phosphate is transferred to the enzyme, forming a phosphoryl- serine residue. 3. Hydrolysis of this phosphate ester by a zinc- bound hydroxide com- pletes the catalytic cycle. This mechanism is supported by studies with chiral phosphate esters (ROP 18 O 17 O 16 O) 2- ; there is no net change in chirality at phoshorus. 1. 2. 3.

35. Principles illustrated: the dimetallic affords hydroxide; the substrate is positioned by residues in the active site; the dimetallic stabilizes the urea leaving group; redox inactive metal; electrostatics

40. PZn(OH 2 ) 2+ PZn(OH) + + H + K eq = 10 -7 M = k f /k r Metallo-  -lactamases, an Emerging Clinical Problem

41. Zn2 Wat2 C181 Wat1 Zn1 H99 H101 H162 D103 H223 N.O. Concha, B.A. Rasmussen, K. Bush, O. Herzberg (1996), Structure 4, 823-836  -Lactamase from Bacteroides fragilis

44. Both mono- and dimetallic centers lower the pK a value of bound water, allowing hydroxide to be delivered at pH 7. Coordination of the leaving group portion of the substrate to a metal ion activates the substrate for nucleophilic attack. Residues not coordinated but in the second coordination sphere can participate directly (serine in phophatases) or indirectly (arginine in alcohol dehydrogenase) in substrate attack, orientation, and/or activation. Carboxylate shifts facilitate substrate binding, activation. Redox inactive metal ions (Zn 2+, Ni 2+, Mn 2+, Co 2+ ) preferred. Summary - Points to Remember