Interprotein and Intraprotein Electron Transfer Mechanisms

Tollin, Gordon

doi:10.1002/9783527618248.ch51

Cited by 29 publications

(21 citation statements)

References 100 publications

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“…The laser flash photolysis apparatus system has been extensively described (49) as has been the basic photochemical process by which 5-deazariboflavin semiquinone (dRFH • ) is generated by reaction between triplet state dRF and the sacrificial reductant and used to reduce redox-active proteins (eqs. 2–5) (50–52).

dRF \to {}^{1}{dRF} \to {}^{3}{dRF}

{}^{3}{dRF} + {AH}_{2} \to dRFH • + AH •

dRFH • + Mo (VI) / Fe (III) \to dRF + Mo + Mo (IV) / Fe (II)

Mo (VI) Fe (II) ⇌_{k_{normalr}}^{k_{normalf}} Mo (V) / Fe (III)

…”

Section: Methodsmentioning

confidence: 99%

Effects of Interdomain Tether Length and Flexibility on the Kinetics of Intramolecular Electron Transfer in Human Sulfite Oxidase

et al. 2010

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Sulfite oxidase (SO) is a vitally important molybdenum enzyme that catalyzes the oxidation of toxic sulfite to sulfate. The proposed catalytic mechanism of vertebrate SO involves two intramolecular one-electron transfer (IET) steps from the molybdenum cofactor to the iron of the integral b-type heme and two intermolecular one-electron steps to exogenous cytochrome c. In the crystal structure of chicken SO (Kisker et al., Cell, 1997, 91, 973-983), which is highly homologous to human SO (HSO), the heme iron and molybdenum centers are separated by 32 Å, and the domains containing these centers are linked by a flexible polypeptide tether. Conformational changes that bring these two centers into closer proximity have been proposed (Feng et al., Biochemistry, 2003, 41, 5816-21) to explain the relatively rapid IET kinetics, which are much faster than theoretically predicted from the crystal structure. In order to explore the proposed role(s) of the tether in facilitating this conformational change, both its length and flexibility were altered in HSO by site-specific mutagenesis and the reactivities of the resulting variants have been studied using laser flash photolysis and steady-state kinetics assays. Increasing the flexibility of the tether by mutating several conserved proline residues to alanines did not produce a discernable systematic trend in the kinetic parameters, although mutation of one residue (P105) to alanine produced a three-fold decrease in the IET rate constant. Deletions of non-conserved amino acids in the 14-residue tether, thereby shortening its length, resulted in more drastically reduced IET rate constants. Thus, the deletion of five amino acid residues decreased IET by 70-fold, so that it was rate-limiting in the overall reaction. The steadystate kinetic parameters were also significantly affected by these mutations, with the P111A mutation causing a five-fold increase in the sulfite K m value, perhaps reflecting a decrease in the ability to bind sulfite. The electron paramagnetic resonance spectra of these Proline to Alanine and deletion mutants are identical to those of wild type HSO, indicating no significant change in the Mo active site geometry.Sulfite oxidase (SO) catalyzes the oxidation of sulfite to sulfate, using oxidized ferricytochrome c (cyt c ox ) as the physiological electron acceptor (eq. 1) (1-4). This reaction is biologically essential, serving as the final step in the catabolism of sulfur containing amino acids, methionine and cysteine, and as a detoxification mechanism for sulfite. † This research was supported by NIH Grant GM-037773 (to JHE); Ruth L. Kirchstein-NIH Fellowship 1F32GM082136-01 (to KJW) *To whom correspondence should be addressed. J.H.E.: jenemark@u.arizona.edu; phone, (520) 621-2245; fax, (520) 626-8065. G.T.:, gtollin@u.arizona.edu; phone, (520) 621-3447; fax, (520) 621-9288. Supporting Information Available: Primer design; iron to molybdenum ratios determined using inductively coupled plasma; and laser flash photolysis results for proline to alanine ...

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dRF \to {}^{1}{dRF} \to {}^{3}{dRF}

{}^{3}{dRF} + {AH}_{2} \to dRFH • + AH •

dRFH • + Mo (VI) / Fe (III) \to dRF + Mo + Mo (IV) / Fe (II)

Mo (VI) Fe (II) ⇌_{k_{normalr}}^{k_{normalf}} Mo (V) / Fe (III)

…”

Section: Methodsmentioning

confidence: 99%

Effects of Interdomain Tether Length and Flexibility on the Kinetics of Intramolecular Electron Transfer in Human Sulfite Oxidase

et al. 2010

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“…Electrostatic interactions between protein/domain surfaces are important in molecular recognition, making the distribution of charged surface amino acids an active determinant of ET kinetics [130, 132]. Pronounced isoform-specific effects of salt concentration on NOS activities have been reported [133], suggesting importance of electrostatic interactions in the NOS electron transfer processes.…”

Section: Mechanism Of the Fmn–heme Iet In Nosmentioning

confidence: 99%

Dissecting regulation mechanism of the FMN to heme interdomain electron transfer in nitric oxide synthases

Feng

Chen

et al. 2014

Journal of Inorganic Biochemistry

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Nitric oxide synthase (NOS), a flavo-hemoprotein, is responsible for biosynthesis of nitric oxide (NO) in mammals. Three NOS isoforms, iNOS, eNOS and nNOS (inducible, endothelial, and neuronal NOS), achieve their biological functions by tight control of interdomain electron transfer (IET) process through interdomain interactions. In particular, the FMN–heme IET is essential in coupling electron transfer in the reductase domain with NO synthesis in the heme domain by delivery of electrons required for O2 activation at the catalytic heme site. Emerging evidence indicates that calmodulin (CaM) activates NO synthesis in eNOS and nNOS by a conformational change of the FMN domain from its shielded electron-accepting (input) state to a new electron-donating (output) state, and that CaM is also required for proper alignment of the FMN and heme domains. In the absence of a structure of full-length NOS, an integrated approach of spectroscopic, rapid kinetics and mutagenesis methods is required to unravel regulation mechanism of the FMN–heme IET process. This is to investigate the roles of the FMN domain motions and the docking between the primary functional FMN and heme domains in regulating NOS activity. The recent developments in this area that are driven by the combined approach are the focuses of this review. A better understanding of the roles of interdomain FMN/heme interactions and CaM binding may serve as a basis for the rational design of new selective modulators of the NOS enzymes.

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“…protein-protein electron transfer rate constants (Meyer et al, , 1987(Meyer et al, , 1993a(Meyer et al, , 1993b(Meyer et al, , 1993cTollin et al, 1984Tollin et al, , 1986Tollin et al, , 1993Przysiecki et al, 1985;Cheddar et al, 1986Cheddar et al, , 1989. In all cases where the experimental data show a monotonic increase or decrease in rate constant with ionic strength, the rate constant versus square root of ionic strength curves can be fit using the parallel plate model monopole term (K;) only.…”

Section: Ja Watkins Et Almentioning

confidence: 99%

A “parallel plate” electrostatic model for bimolecular rate constants applied to electron transfer proteins

et al. 1994

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A "parallel plate" model describing the electrostatic potential energy of protein-protein interactions is presented that provides an analytical representation of the effect of ionic strength on a bimolecular rate constant. The model takes into account the asymmetric distribution of charge on the surface of the protein and localized charges at the site of electron transfer that are modeled as elements of a parallel plate condenser. Both monopolar and dipolar interactions are included. Examples of simple (monophasic) and complex (biphasic) ionic strength dependencies obtained from experiments with several electron transfer protein systems are presented, all of which can be accommodated by the model. The simple cases do not require the use of both monopolar and dipolar terms (i.e., they can be fit well by either alone). The biphasic dependencies can be fit only by using dipolar and monopolar terms of opposite sign, which is physically unreasonable for the molecules considered. Alternatively, the high ionic strength portion of the complex dependencies can be fit using either the monopolar term alone or the complete equation; this assumes a model in which such behavior is a consequence of electron transfer mechanisms involving changes in orientation or site of reaction as the ionic strength is varied. Based on these analyses, we conclude that the principal applications of the model presented here are to provide information about the structural properties of intermediate electron transfer complexes and to quantify comparisons between related proteins or site-specific mutants. We also conclude that the relative contributions of monopolar and dipolar effects to protein electron transfer kinetics cannot be evaluated from experimental data by present approximations.

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Interprotein and Intraprotein Electron Transfer Mechanisms

Cited by 29 publications

References 100 publications

Effects of Interdomain Tether Length and Flexibility on the Kinetics of Intramolecular Electron Transfer in Human Sulfite Oxidase

Effects of Interdomain Tether Length and Flexibility on the Kinetics of Intramolecular Electron Transfer in Human Sulfite Oxidase

Dissecting regulation mechanism of the FMN to heme interdomain electron transfer in nitric oxide synthases

A “parallel plate” electrostatic model for bimolecular rate constants applied to electron transfer proteins

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