Principles of Bioinorganic Chemistry - 2004

Principles of Bioinorganic Chemistry - 2004Note: 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.

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_1030867717.doc

Lecture

Date

Lecture Topic

Reading

Problems

1

9/9 (Th)

Intro; Choice, Uptake, Assembly of Mn+ Ions

Ch. 5

Ch. 1

2

9/14 (Tu)

Metalloregulation of Gene Expression

Ch. 6

Ch. 2

3

9/16 (Th)

Metallochaperones; Mn+-Folding, X-linking

Ch. 7

Ch. 3

4

9/21 (Tu)

Med. Inorg. Chem./Metalloneurochemistry

Ch. 8

Ch. 4

5

9/23 (Th)

Mssbauer, EPR, IR Spectral Fundamentals

Ch. 9

Ch. 5

6

9/28 (Tu)

Electron Transfer; Fundamentals

Ch. 9

Ch. 6

7

9/30 (Th)

Long-Distance Electron Transfer

Ch. 10

Ch. 7

8

10/5 (Tu)

Hydrolytic Enzymes, Zinc, Ni, Co

Ch. 10

9

10/7 (Th)

CO and Bioorganometallic Chemistry

TBA

Ch. 8

10

10/12 (Tu)

Dioxygen Carriers: Hb, Mb, Hc, Hr

Ch. 11

Ch. 9

11

10/14 (Th)

O2 Activation, Hydroxylation: MMO, ToMO

Ch. 11

Ch. 10

12

10/19 (Tu)

Model Chemistry for O2 Carriers/Activators

Ch. 12

Ch. 11

13

10/21 (Th)

Complex Systems: cyt. oxidase; nitrogenase

Ch. 12

Ch. 12

14

TBA

Term Examination

_1031489789.doc

Lecture

Date

Lecture Topic

Reading

Problems

1

9/9 (Th)


Ch. 5

Ch. 1

2

9/14 (Tu)


Ch. 6

Ch. 2

3

9/16 (Th)


Ch. 7

Ch. 3

4

9/21 (Tu)


Ch. 8

Ch. 4

5

9/23 (Th)


Ch. 9

Ch. 5

6

9/28 (Tu)


Ch. 9

Ch. 6

7

9/30 (Th)


Ch. 10

Ch. 7

8

10/5 (Tu)


Ch. 10

9

10/7 (Th)


TBA

Ch. 8

10

10/12 (Tu)


Ch. 11

Ch. 9

11

10/14 (Th)


Ch. 11

Ch. 10

12

10/19 (Tu)


Ch. 12

Ch. 11

13

10/21 (Th)


Ch. 12

Ch. 12

14

TBA

Term Examination

_1032010444.doc

Lecture

Date

Lecture Topic

Reading

Problems

1

9/9 (Th)


Ch. 5

Ch. 1

2

9/14 (Tu)


Ch. 6

Ch. 2

3

9/16 (Th)


Ch. 7

Ch. 3

4

9/21 (Tu)


Ch. 8

Ch. 4

5

9/23 (Th)


Ch. 9

Ch. 5

6

9/28 (Tu)


Ch. 9

Ch. 6

7

9/30 (Th)


Ch. 10

Ch. 7

8

10/5 (Tu)


Ch. 10

9

10/7 (Th)


TBA

Ch. 8

10

10/12 (Tu)


Ch. 11

Ch. 9

11

10/14 (Th)


Ch. 11

Ch. 10

12

10/19 (Tu)


Ch. 12

Ch. 11

13

10/21 (Th)


Ch. 12

Ch. 12

14

TBA

Term Examination

_1032607554.doc

Lecture

Date

Lecture Topic

Reading

Problems

1

9/9 (Th)


Ch. 5

Ch. 1

2

9/14 (Tu)


Ch. 6

Ch. 2

3

9/16 (Th)


Ch. 7

Ch. 3

4

9/21 (Tu)


Ch. 8

Ch. 4

5

9/23 (Th)


Ch. 9

Ch. 5

6

9/28 (Tu)


Ch. 9

Ch. 6

7

9/30 (Th)


Ch. 10

Ch. 7

8

10/5 (Tu)


Ch. 10

9

10/7 (Th)


TBA

Ch. 8

10

10/12 (Tu)


Ch. 11

Ch. 9

11

10/14 (Th)


Ch. 11

Ch. 10

12

10/19 (Tu)


Ch. 12

Ch. 11

13

10/21 (Th)


Ch. 12

Ch. 12

14

11/4 (Th)

Term Examination

_1032873579.doc

Lecture

Date

Lecture Topic

Reading

Problems

1

9/9 (Th)


Ch. 5

Ch. 1

2

9/14 (Tu)


Ch. 6

Ch. 2

3

9/16 (Th)


Ch. 7

Ch. 3

4

9/21 (Tu)


Ch. 8

Ch. 4

5

9/23 (Th)


Ch. 9

Ch. 5

6

9/28 (Tu)


Ch. 9

Ch. 6

7

9/30 (Th)


Ch. 10

Ch. 7

8

10/5 (Tu)


Ch. 10

9

10/7 (Th)


TBA

Ch. 8

10

10/12 (Tu)


Ch. 11

Ch. 9

11

10/14 (Th)


Ch. 11

Ch. 10

12

10/19 (Tu)


Ch. 12

Ch. 11

13

10/21 (Th)


Ch. 12

Ch. 12

14

11/4 (Th)

Term Examination

_1032010640.doc

Lecture

Date

Lecture Topic

Reading

Problems

1

9/9 (Th)


Ch. 5

Ch. 1

2

9/14 (Tu)


Ch. 6

Ch. 2

3

9/16 (Th)


Ch. 7

Ch. 3

4

9/21 (Tu)


Ch. 8

Ch. 4

5

9/23 (Th)


Ch. 9

Ch. 5

6

9/28 (Tu)


Ch. 9

Ch. 6

7

9/30 (Th)


Ch. 10

Ch. 7

8

10/5 (Tu)


Ch. 10

9

10/7 (Th)


TBA

Ch. 8

10

10/12 (Tu)


Ch. 11

Ch. 9

11

10/14 (Th)


Ch. 11

Ch. 10

12

10/19 (Tu)


Ch. 12

Ch. 11

13

10/21 (Th)


Ch. 12

Ch. 12

14

11/4 (Th)

Term Examination

_1031489813.doc

Lecture

Date

Lecture Topic

Reading

Problems

1

9/9 (Th)


Ch. 5

Ch. 1

2

9/14 (Tu)


Ch. 6

Ch. 2

3

9/16 (Th)


Ch. 7

Ch. 3

4

9/21 (Tu)


Ch. 8

Ch. 4

5

9/23 (Th)


Ch. 9

Ch. 5

6

9/28 (Tu)


Ch. 9

Ch. 6

7

9/30 (Th)


Ch. 10

Ch. 7

8

10/5 (Tu)


Ch. 10

9

10/7 (Th)


TBA

Ch. 8

10

10/12 (Tu)


Ch. 11

Ch. 9

11

10/14 (Th)


Ch. 11

Ch. 10

12

10/19 (Tu)


Ch. 12

Ch. 11

13

10/21 (Th)


Ch. 12

Ch. 12

14

TBA

Term Examination

_1030867800.doc

Lecture

Date

Lecture Topic

Reading

Problems

1

9/9 (Th)


Ch. 5

Ch. 1

2

9/14 (Tu)


Ch. 6

Ch. 2

3

9/16 (Th)


Ch. 7

Ch. 3

4

9/21 (Tu)


Ch. 8

Ch. 4

5

9/23 (Th)


Ch. 9

Ch. 5

6

9/28 (Tu)


Ch. 9

Ch. 6

7

9/30 (Th)


Ch. 10

Ch. 7

8

10/5 (Tu)


Ch. 10

9

10/7 (Th)


TBA

Ch. 8

10

10/12 (Tu)


Ch. 11

Ch. 9

11

10/14 (Th)


Ch. 11

Ch. 10

12

10/19 (Tu)


Ch. 12

Ch. 11

13

10/21 (Th)


Ch. 12

Ch. 12

14

TBA

Term Examination

_1031474426.doc

Lecture

Date

Lecture Topic

Reading

Problems

1

9/9 (Th)


Ch. 5

Ch. 1

2

9/14 (Tu)


Ch. 6

Ch. 2

3

9/16 (Th)


Ch. 7

Ch. 3

4

9/21 (Tu)


Ch. 8

Ch. 4

5

9/23 (Th)


Ch. 9

Ch. 5

6

9/28 (Tu)


Ch. 9

Ch. 6

7

9/30 (Th)


Ch. 10

Ch. 7

8

10/5 (Tu)


Ch. 10

9

10/7 (Th)


TBA

Ch. 8

10

10/12 (Tu)


Ch. 11

Ch. 9

11

10/14 (Th)


Ch. 11

Ch. 10

12

10/19 (Tu)


Ch. 12

Ch. 11

13

10/21 (Th)


Ch. 12

Ch. 12

14

TBA

Term Examination

_1030867766.doc

Lecture

Date

Lecture Topic

Reading

Problems

1

9/9 (Th)


Ch. 5

Ch. 1

2

9/14 (Tu)


Ch. 6

Ch. 2

3

9/16 (Th)


Ch. 7

Ch. 3

4

9/21 (Tu)


Ch. 8

Ch. 4

5

9/23 (Th)


Ch. 9

Ch. 5

6

9/28 (Tu)


Ch. 9

Ch. 6

7

9/30 (Th)


Ch. 10

Ch. 7

8

10/5 (Tu)


Ch. 10

9

10/7 (Th)


TBA

Ch. 8

10

10/12 (Tu)


Ch. 11

Ch. 9

11

10/14 (Th)


Ch. 11

Ch. 10

12

10/19 (Tu)


Ch. 12

Ch. 11

13

10/21 (Th)


Ch. 12

Ch. 12

14

TBA

Term Examination

_998891506.doc

Lecture

Date

Lecture Topic

Reading

Problems

1

9/4 (Th)


Ch. 5

Ch. 1

2

9/ 9 (Tu)


Ch. 6

Ch. 2

3

9/11 (Th)

Metallochaperones; Metal Folding, X-linking

Ch. 7

Ch. 3

4

9/ 16 (Tu)

Zinc Fingers; Metal Folding; Cisplatin

Ch. 8

Ch. 4

5

9/18 (Th)


Ch. 9

Ch. 5

6

9/23 (Tu)


Ch. 9

Ch. 6

7

9/25 (Th)


Ch. 10

Ch. 7

8

9/30 (MU)

Model Complexes for Metallohydrolases

Ch. 10

9

10/2 (MU)


Ch. 11

10

10/7 (Tu)

O2 Activation, Hydroxylation: MMO, P-450, R2

Ch. 11

Ch. 8

11

10/9 (Th)


Ch. 11

Ch. 9

12

10/16 (Th)


Ch. 12

Ch. 10

13

10/21 (Tu)

Metalloneurochemistry/Medicinal Inorg. Chem.

Ch. 12

Ch. 11

14

10/23 (Th)

Term Examination

Ch. 12

Ch. 12

_1030771764.doc

Lecture

Date

Lecture Topic

Reading

Problems

1

9/9 (Th)


Ch. 5

Ch. 1

2

9/14 (Tu)


Ch. 6

Ch. 2

3

9/16 (Th)


Ch. 7

Ch. 3

4

9/21 (Tu)


Ch. 8

Ch. 4

5

9/23 (Th)


Ch. 9

Ch. 5

6

9/28 (Tu)


Ch. 9

Ch. 6

7

9/30 (Th)


Ch. 10

Ch. 7

8

10/5 (Tu)


Ch. 10

9

10/7 (Th)


TBA

Ch. 8

10

10/12 (Tu)


Ch. 11

Ch. 9

11

10/14 (Th)


Ch. 11

Ch. 10

12

10/19 (Tu)


Ch. 12

Ch. 11

13

10/21 (Th)


Ch. 12

Ch. 12

14

TBA

Term Examination

_998891435.doc

Lecture

Date

Lecture Topic

Reading

Problems

1

9/4 (Th)


Ch. 5

Ch. 1

2

9/ 9 (Tu)


Ch. 6

Ch. 2

3

9/ 11 (Th)

Metallochaperones; Metal Folding, X-linking

Ch. 7

Ch. 3

4

9/16 (Tu)

Metals in Medicine; Cisplatin

Ch. 8

Ch. 4

5

9/18 (Th)


Ch. 9

Ch. 5

6

9/23 (Tu)


Ch. 9

Ch. 6

7

9/25 (Th)


Ch. 10

Ch. 7

8

9/30 (MU)

Model Complexes for Metallohydrolases

Ch. 10

9

10/2 (MU)


Ch. 11

10

10/7 (Tu)

O2 Activation, Hydroxylation: MMO, P-450, R2

Ch. 11

Ch. 8

11

10/9 (Th)


Ch. 11

Ch. 9

12

10/16 (Th)


Ch. 12

Ch. 10

13

10/21 (Tu)

Metalloneurochemistry/Medicinal Inorg. Chem.

Ch. 12

Ch. 11

14

10/23 (Th)

Term Examination

Ch. 12

Ch. 12

Dioxygen Carriers: Hb, Mb, Hc, HrExamples of Atom- and Group-Transfer ChemistryPRINCIPLES:Both substrate binding and redox changes occurCoupled proton-electron transfer steps set the redox potentialsClosely positioned redox/acid-base units work in concertInteractions with substrates/other proteins gate electron transferTwo-electron transfer strategies include 2 metals, M-porphyrinsMetal centers used to create or destroy radical speciesChanges in metal coordination spheres can facilitate allosteryBioinorganic chemistry of dioxygen paramount exampleILLUSTRATIONS:

O2 Binding and Transport: hemoglobin (Hb), myoglobin (Mb), hemocyanin (Hc), and hemerythrin (Hr)O2 Activation: cytochrome P-450, tyrosinase, methane monooxygenase; dioxygenases

Properties of Protein Dioxygen Carriers

Property

Hemoglobin

Hemerythrin

Hemocyanin

Metal

Fe

Fe

Cu

Ox. state of metal

in deoxy protein

(II)

(II)

(I)

Metal:O2

Fe:O2

2Fe:O2

2Cu:O2

Color, oxygenated

Red

Violet-pink

Blue

Color,

deoxygenated

Red-purple

Colorless

Colorless

Metal coordination

Porphyrin

ring

Protein side chains

Protein side chains

Molecular Weight

65,000

108,000

400,000 to 20,000,000

Number of

subunits

4

8

Many

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

Vibrational Spectroscopic Evidence that OxyHb and OxyMb are Formally FeIIIO2- SpeciesFrom resonance Raman spectroscopy the OO stretchin oxyMb is measured to be ~ 1105 cm-1. The protein is also diamagnetic (d5, Fe(III) and O2- couple).

Structural and Spin State Changes upon Binding of Dioxygen to an Iron Porphyrin CenterHigh-spinferrousLow-spinferricDeoxy 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 MbThe problem:FeIIP + O2 FeIIIPO2- PFeIIIO OFeIIIP....2PFeIV=O: PFeIIIOFeIIIP..FeIIPm-oxo, dimerThe 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. FeIIPferryl

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

Structure of AzidomethemerythrinContains 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.

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 nOO, 844 cm-1 in the terminally bound peroxide region. nFeOFe, 486 cm-1, resonance enhanced symmetric stretch. The asymmetric stretch occurs at 757 cm-1.Mixed-valent, semimet Hr, Fe(II)Fe(III): inactive.

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

Early Structural Models for MethemerythrinThese 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.

Early Structural Models for DeoxyhemerythrinNone does the chemistry of the protein!

Properties of Oxy Hr, Deoxy Hr, and Models

Dioxygen Binding Chemistry of the Hr Model ComplexProduct matches protein Mssbauer, resonance Raman, UV-vis spectra

Hemocyanins - Dicopper Dioxygen CarriersProperties: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 nOO, 745-750 cm-1 in the peroxide region, but low.

Unusual structure, first established by model chemistry: OCu Cu O

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

...

Schematic Views of Deoxy and Oxy HcNote, Type III copper

Model Chemistry for Deoxy and Oxy HcKarlin modelKitajima model

Important RelationshipsReversible 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??

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

Principles Illustrated by these CasesSubstrate 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.

The Mineral Springs in Bath, England,Source of Methylococcus capsulatus (Bath)The Restitutive Contents of the WATERs 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!

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)

Evolutionary Relationships Between Multicomponent MonooxygenasesCommon AncestorAmo Alkene MonooxygenaseSoluble Methane MonooxygenasesFour Component Alkene/Aromatic MonooxygenasesPhenol HydroxylasesMethane MonooxygenaseButane MonooxygenaseToluene MonooxygenasesPhenol HydroxylasesIsoprene MonooxygenasesDimethyl Sulfde Hydroxylase Phenol Hydroxylase

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

Properties of MethanotrophspMMO, CusMMO, 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.

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 catalysisGlobal Research Goals

Soluble Methane Monooxygenase (sMMO) Component Structures & Reaction Cycle

sMMO is a Multicomponent Enzyme

Reactions of CH3X Substrates with QMMOHQ Mssbauer (d 0.17 mm s-1)UV-vis, 420 nm; EXAFSCH4CH3OHH+

Mechanism for Methanol FormationGherman, 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/molMethaneQ

First Electron Transfer for Methanol Formation17.9 kcal/mol First electron transfer occurs here and determines the barrier height; one Fe reduced to Fe(III) as OH bond forms.

Mechanism for Methanol FormationThis transition state is 1.3 kcal/mol uphill from the boundradical intermediate, affording a rate constant in accordwith most radical clock substrate probe studies.

Electronic Details of Second Electron TransferDonor OrbitalBound MethylRadical (b-Spin)Acceptor OrbitalFe1 d-(x2-y2)(b-Spin-LUMO)Mediator OrbitalOxo p(z)(doubly occupied)Baik, Gherman, Friesner & Lippard, J. Am. Chem. Soc. 2002, 124, 14608.

Electronic Details of Second Electron TransferHO rotation promotes intramolecularb-electron transfer from the oxo lone pairorbital to the metal-based LUMO.The remaining radicaloid a-electron onthe bridging oxo group has the correctspin to recombine with the b-electron on the substrate to form a s-bond.

Overall Energetics and Methanol Release1.3-69.7E in kcal/mol

Reactions of Q with Substrates Reveal ComplexitiesCH4CD4C2D6Puzzles: This result indicates that, for ethane, the rate-determining step in not CH 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 CH bond activation energy by ~5.6 kcal/mol, from both experiment and theory. C2H6

kobs 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, CH3NO2 Open circles CD3NO2

pH = 7, 20 C ; KIE, 8.1

Single Turnover of Q with Nitromethane-d3 at 25Cby Stopped-Flow Infrared SpectroscopyLoss of Q monitored by stopped-flow spectrophotometry at 420 nm; kobs 0.39 s-1Loss of nitromethane-d3 monitored by stopped-flow IR spectroscopy at 1548 cm-1; kobs 0.39 s-1Muthusamy, 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

KIE for Reactions of Q with CH3X SubstratesaCLASS I SUBSTRATESH atom abstraction rate-determining:CH4, CH3CN, CH3NO2

CLASS II SUBSTRATESBinding rate-determining: C2H6, CH3OH

Ambundo, E. A.; Friesner, R. A.; Lippard, S. J. J. Am. Chem. Soc. 2002, 124, 8770-8771.

Substrate

KIE, kH/kD

CH4/CD4

23.1 1.1

C2H6/C2D6

1.00 0.04

CH3CN/CD3CN

46.4 2.3

CH3NO2/CD3NO2

8.1 0.2

CH3OH/CD3OH

1.01 0.01

apH = 7, 20 (C.

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

Reactions of Substrates with HperoxoMMOHperoxoMssbauer (d 0.66 mm s-1)UV-vis, 725, 410 nm

Preliminary Evidence for Hperoxo Reacting with SubstratesHperoxo appears to react with propyleneLow solubility of substrates limits experimentCould 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.open circles methanefilled circles - propylene

Hperoxo and Q Reactions with Ethyl Vinyl EtherThe product of propylene reaction with Hperoxo is propylene oxide

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

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

Substrate Access in MMOH and ToMOH

Toluene /o-Xylene Monooxygenase from Pseudomonas stutzeri OX1FeFeRieskeregulationToMOBToMORFe2S2ToMOHe-e-+ O2+ H2OAll components are expressed recombinantly in E. coli!ToMO has relaxed substrate specificitySubstrates:toluene, xylenes, benzene, napthalene, phenol, cresols, ethylbenzene, styrene, halogenated ethylenesFeFeCarfaro, V., et. al. Eur. J. Biochem. 2002, 296, 5689-5699.

M. H. Sazinsky, J. Bard, A. Di Donato, & S. J. Lippard, J. Biol. Chem. 2004, 279, 30600-30610.Crystallization of ToMOHCrystal Facts:

Space group P3121

Unit cell () 182 x 182 x 67

Conditions: ToMOB NaN3 (antimicrobial)2Fo-Fc map from SAD Phasing0.7 mm

Crystal Structures of ToMOH and MMOHThe 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. MMOHToMOHg-subunits

ToMOHox + thioglycolateActive Site Structures of ToMOHox and MMOHoxMMOHox + acetateToMOH has OH- and RCO2- bridges similar to those of MMOH and T4MOH.Pikus, J. D. et. al. Biochemistry. (1996), 35, 9106-9119.Cadieux, E., et al. Biochemistry. (2002), 41, 10680-10691. Fe-Fe distance 3.0Fe-Fe distance 3.0

Spectroscopy of HoxT4MOHMMOHPHAbsorption maxima (nm)Mssbauer , EQ(mm/s)2800.51, 0.93 (85%)0.56, 1.55 (15%)2800.52, 1.16 0.51, 0.91 280, 3500.54, 1.73 (85%)0.48, 0.79 (15%)

ToMOH has a Surface Accessible Channel Channel is 35-40 long, 6-10 wide Big enough for substrates and products Diiron center has direct access to surface

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 channelUniversal Pathways for Small Molecule AccessCH2Br2 in MMOH cavities 2 and 3

Substrate Access at the Diiron CenterThe 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, 141-152TomoHMMOHThe ToMOH Fe2 center is more accessible to solvent.

Electron/H+ Transfer in MMOH and ToMOH

Testing the Role of an Absolutely Conserved AsparagineHox + BromoethanolHredAsn 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?

Steady-State Turnover of WT and N202A ToMOHSingle turnover results for ToMOHred + O2 + 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))

Principles of Bioinorganic Chemistry - 2004

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