Control of heme protein redox potential and reduction rate: linear free energy relation between potential and ferric spin state equilibrium

Fisher, Mark T.; Sligar, Stephen G.

doi:10.1021/ja00303a045

Cited by 174 publications

(138 citation statements)

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“…For example, dimerization may change the reactivity of the heme iron in the full-length subunit such that it can accept electrons from its reductase domain. Indeed, our related work (22,24) indicates that the heme iron in an iNOS monomer is exposed to solvent and as a result is predominantly six-coordinate low spin, which in cytochrome P-450 is associated with a decrease in heme iron reduction potential (29,30). Dimerization, which shifts the iNOS heme iron spin equilibrium back toward high spin (22,24), could thus enable electron transfer by increasing the reduction potential of the heme iron in the full-length subunit.…”

Section: Characteristics Of a No Synthase Heterodimer 7311mentioning

confidence: 97%

Heme Iron Reduction and Catalysis by a Nitric Oxide Synthase Heterodimer Containing One Reductase and Two Oxygenase Domains

Siddhanta

Abu-Soûd

et al. 1996

Journal of Biological Chemistry

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Inducible nitric oxide (NO) synthase (iNOS) is com-prised of an oxygenase domain containing heme, tetrahydrobiopterin, the substrate binding site, and a reductase domain containing FAD, FMN, calmodulin, and the NADPH binding site. Enzyme activity requires a dimeric interaction between two oxygenase domains with the reductase domains attached as monomeric extensions. To understand how dimerization activates iNOS, we synthesized an iNOS heterodimer comprised of one fulllength subunit and one histidine-tagged subunit that was missing its reductase domain. The heterodimer was purified using nickel-Sepharose and 2,5-ADP affinity chromatography. The heterodimer catalyzed NADPHdependent NO synthesis from L-arginine at a rate of 52 ؎ 6 nmol of NO/min/nmol of heme, which is half the rate of purified iNOS homodimer. Heterodimer NO synthesis was associated with reduction of only half of its heme iron by NADPH, in contrast with near complete heme iron reduction in an iNOS homodimer. Full-length iNOS monomer preparations could not synthesize NO nor catalyze NADPH-dependent heme iron reduction. Thus, dimerization activates NO synthesis by enabling electrons to transfer between the reductase and oxygenase domains. Although a single reductase domain can reduce only one of two hemes in a dimer, this supports NO synthesis from L-arginine.

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Section: Characteristics Of a No Synthase Heterodimer 7311mentioning

confidence: 97%

Heme Iron Reduction and Catalysis by a Nitric Oxide Synthase Heterodimer Containing One Reductase and Two Oxygenase Domains

Siddhanta

Abu-Soûd

et al. 1996

Journal of Biological Chemistry

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“…Martin and co-workers 77,298 underlined another discrepancy with solution studies: apart from an early work with P450cam adsorbed on graphite, 316 electrochemical investigations with many P450 enzymes cast into DDAB films did not detect the positive shift in reduction potential upon binding of substrate that is the basis of the regulation mechanism proposed by Gunsalus and coworkers [317][318][319][320] and explains why selective substrate oxidation occurs in preference to O 2 reduction. It has been proposed that this inconsistency may result from DDAB molecules blocking access to the substrate-binding cavity 302 or from partial unfolding induced by the immobilization and electric field effects, as occurs on various Ag/SAM electrodes.…”

Section: Observing Reasonable Noncatalytic Signalsmentioning

confidence: 99%

Direct Electrochemistry of Redox Enzymes as a Tool for Mechanistic Studies

2008

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“…Thus, the kinetic traces at Pd concentrations higher than 2 M were employed for the analysis. (18). In the latter case, k and k 0 correspond to the reduction rate of P450 cam bound to substrate analogs and d-camphor, respectively.…”

Section: Table IImentioning

confidence: 99%

Role of Arg112 of Cytochrome P450cam in the Electron Transfer from Reduced Putidaredoxin

Unno

Shimada

Toba

et al. 1996

Journal of Biological Chemistry

100

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The mechanism for the reduction of ferric cytochrome P450 cam by reduced putidaredoxin, the physiological electron donor for the cytochrome, has been studied by using site-directed mutants of cytochrome P450 cam , in which Arg 112, an amino acid residue at the presumed binding site for putidaredoxin, was changed to several other amino acid residues. Cytochrome P450 is a generic name given to a family of protoheme-proteins that metabolize a wide variety of natural and unnatural substances such as steroids, fatty acids, hydrocarbons, and xenobiotics. Among them, cytochrome P450 cam (P450 cam ) 1 of Pseudomonas putida (CYP101) has been the focus of intense mechanistic studies; its three-dimensional structure has been solved at 1.63-Å resolution by Poulos and co-workers (1, 2), allowing us to make detailed analyses of the structure-function relationship. It catalyzes the hydroxylation of d-camphor to give 5-exo-hydroxycamphor as in the following scheme.The monooxygenation reaction requires, in addition to dcamphor and molecular oxygen, two reducing equivalents, which are transferred from NADH to P450 cam through a specific electron transfer system composed of NADH-putidaredoxin reductase (PdR), a flavoprotein, and putidaredoxin (Pd), an iron-sulfur (Fe 2 S 2 ) protein. In the reaction, Pd receives electrons from PdR and transfers them to P450 cam ; Pd serves as the direct electron donor for P450 cam .When the mechanism for the electron transfer reaction between Pd and P450 cam was examined, reduced Pd and ferric P450 cam molecules were found to associate rapidly to form a bimolecular complex, followed by an intracomplex electron transfer giving ferrous P450 cam and oxidized Pd (3-5). Then, on the basis of results obtained from computer modeling of P450 cam -cytochrome b 5 complex, the binding site of Pd on P450 cam was suggested (6) to be in the proximal surface of P450 cam , which contains four basic amino acid residues (Arg , another basic amino acid among the four basic residues, evoked a dramatic decrease in the catalytic activity of P450 cam . Catalytic activities of Cys, Glu, and Gln mutants were less than 1% of that of the wild-type enzyme in a reconstituted assay system composed of NADH, PdR, Pd, and a mutant P450 cam . Thus, the presumed binding site in the proximal surface could be the real site for the binding of Pd to P450 cam at least in part.In the present study, we further elucidate the role of Arg 112 in the electron transfer reaction from reduced Pd to ferric P450 cam by using site-directed mutants of P450 cam , in which the Arg residue was changed to a Lys, Cys, Tyr, or Met residue. Since they have different charges and hydrogen-bonding capacities with one another, we had hoped that their use might give us a clue to identify the role of the basic residue Arg 112 in the Pd-P450 cam interaction. The results revealed that the oxidation and reduction (redox) potential of the heme iron in P450 cam was * This work was supported in part by Grants-in-aid from the Ministry of Education, Science, and Culture...

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Control of heme protein redox potential and reduction rate: linear free energy relation between potential and ferric spin state equilibrium

Cited by 174 publications

References 0 publications

Heme Iron Reduction and Catalysis by a Nitric Oxide Synthase Heterodimer Containing One Reductase and Two Oxygenase Domains

Heme Iron Reduction and Catalysis by a Nitric Oxide Synthase Heterodimer Containing One Reductase and Two Oxygenase Domains

Direct Electrochemistry of Redox Enzymes as a Tool for Mechanistic Studies

Role of Arg112 of Cytochrome P450cam in the Electron Transfer from Reduced Putidaredoxin

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