Kinetics of flavin semiquinone reduction of the components of the cytochrome c-cytochrome b5 complex

Eltis, Lindsay D.; Ag, Mauk; Jt, Hazzard; Ma, Chunyue; Tollin, Gordon

doi:10.1021/bi00415a011

Cited by 14 publications

(6 citation statements)

References 26 publications

(56 reference statements)

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“…In fact, the most recent measurements, using a ruthenium Rogers & Sligar, 1991;Qin et al, 1991). Likewise, steady-state kinetic measurements of cytochrome c mutants and a comparison of the kinetics of reduction of native and dimethyl-esterified heme propionate cytochrome ¿5 (Eltis et al, 1988;Whitford et al, 1991;Burrows et al, 1991) were in apparent agreement with the model. Previous investigations of interprotein electron transfer between cytochrome ¿5 and cytochrome c have utilized the water-soluble domain of microsomal cytochrome ¿5, derived either from trypsin cleavage of the membrane tail of the bovine liver protein Mauk et al, 1986;McLendon & Miller, 1986;Wendoloski et al, 1987;Eltis et al, 1991) or from the recombinant rat liver cytochrome ¿5 obtained from gene synthesis and bacterial expression of the water-soluble fragment that would have been obtained upon trypsin cleavage of the rat liver microsomal protein (Bodman et al, 1986;Rogers et al, 1988;Rogers & Sligar, 1991;Qin et al, 1991;Willie et al, 1992).…”

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confidence: 54%

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Laser flash photolysis studies of electron transfer to the cytochrome b5-cytochrome c complex

Rivera²,

Fa³

et al. 1993

Biochemistry

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Rate constants for electron transfer in the complex between recombinant rat mitochondrial outer membrane cytochrome b5 or the tryptic fragment of bovine liver cytochrome b5 and horse mitochondrial cytochrome c were measured by laser flash photolysis of 5-deazariboflavin-EDTA solutions. When an excess of cytochrome b5 was titrated with increasing amounts of cytochrome c at low ionic strength and electron transfer was initiated by a laser flash, both proteins were rapidly reduced by deazariboflavin semiquinone. The initial photoreduction was followed by a slower second-order reduction of b5 complexed oxidized cytochrome c by free reduced cytochrome b5. At an 8:1 ratio of cytochromes b5 to c, the pseudo-first-order rate constant for reduction of complexed cytochrome c increased 3-5-fold between ionic strengths of 5 and 40 mM, and then dropped precipitously at higher ionic strengths. The ionic strength dependent increase in rate constant is likely to be due to relief of steric hindrance via rearrangement of cytochrome c in the complex. The reaction rate showed no sign of saturation at any ionic strength, indicating a first-order rate constant greater than 10(4) s-1 within a transient ternary protein complex; i.e., interprotein electron transfer approaches the largest values previously reported for the stable binary protein complex (approximately 4 x 10(5) s-1). Our results emphasize the flexibility of electron-transfer protein complexes, which had previously been modeled in a single conformation with specific salt bridges. It appears that a variety of orientations can exist within such protein-protein complexes and that the population of conformations changes with ionic strength.(ABSTRACT TRUNCATED AT 250 WORDS)

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confidence: 54%

“…The laser flash photolysis apparatus and methods of data collection and analysis were as previously described (Tollin et al, 1986;Eltis et al, 1988; . A laser flash at 400 nm excites 5-deazariboflavin to the triplet state, which in turn oxidizes EDTA, resulting in formation of 5-deazariboflavin semiquinone in less than 1 /is.…”

Section: Methodsmentioning

confidence: 99%

Laser flash photolysis studies of electron transfer to the cytochrome b5-cytochrome c complex

Rivera²,

Fa³

et al. 1993

Biochemistry

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“…Chemical modification of the cytochrome c amino groups on decreases the rate of reaction with cytochrome bs, supporting their involvement in binding (Ng et al, 1977;Stonehuerner et al, 1979;Smith et al, 1980). Mauk et al (1982) obtained direct spectroscopic evidence for the formation of a 1:1 complex at low ionic strength and found that methyl esterification of the cytochrome bs heme propionate alters the orientation of the complex (Reid et al, 1984;Mauk et al, 1986;Eltis et al, 1988). Rodgers et al (1988 and Rodgers and Sligar (1991) used site-directed mutagenesis to change specific cytochrome bs carboxylate groups to the corresponding amides, resulting in decreases in binding strength and specific volume changes which map the interaction domain to that proposed by Salemme (1976).…”

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confidence: 91%

Genetic engineering of redox donor sites: measurement of intracomplex electron transfer between ruthenium-65-cytochrome b5 and cytochrome c

Willie¹,

Stayton²,

Sligar³

et al. 1992

Biochemistry

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The de novo design and synthesis of ruthenium-labeled cytochrome b5 that is optimized for the measurement of intracomplex electron transfer to cytochrome c are described. A single cysteine was substituted for Thr-65 of rat liver cytochrome b5 by recombinant DNA techniques [Stayton, P. S., Fisher, M. T., & Sligar, S. G. (1988) J. Biol. Chem. 263, 13544-13548]. The single sulfhydryl group on T65C cytochrome b5 was then labeled with [4-(bromomethyl)-4'-methylbipyridine] (bisbipyridine)ruthenium2+ to form Ru-65-cyt b5. The ruthenium group at Cys-65 is only 12 A from the heme group of cytochrome b5 but is not located at the binding site for cytochrome c. Laser excitation of the complex between Ru-65-cyt b5 and cytochrome c results in electron transfer from the excited state Ru(II*) to the heme group of Ru-65-cyt b5 with a rate constant greater than 10(6) s-1. Subsequent electron transfer from the heme group of Ru-65-cyt b5 to the heme group of cytochrome c is biphasic, with a fast-phase rate constant of (4 +/- 1) x 10(5) s-1 and a slow-phase rate constant of (3 +/- 1) x 10(4) s-1. This suggests that the complex can assume two different conformations with different electron-transfer properties. The reaction becomes monophasic and the rate constant decreases as the ionic strength is increased, indicating dissociation of the complex.(ABSTRACT TRUNCATED AT 250 WORDS)

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“…This study was hindered for a long time by the temporal discrepancy between the slowness of mechanical mixing devices (few milliseconds) with respect to the submillisecond rates of intraenzymic electron transfer. This limitation has recently been circumvented by the introduction of photodriven redox perturbation techniques initiated by photoexcitation of flavines or ruthenium complexes to their triplet states (1)(2)(3)(4)(5)(6). These reactions were instrumental in the pulse reduction of cytochromes or cytochrome complexes.…”

Section: Introductionmentioning

confidence: 99%

Fast Redox Perturbation of Aqueous Solution by Photoexcitation of Pyranine

Kotlyar

Borovok

Raviv

et al. 1996

Photochem & Photobiology

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Intense illumination (60-120 MW/cm2) of an oxygen-free aqueous solution of pyranine (8-hydroxypyrene-l,3,64risulfonate) by the third harmonic frequency of an Nd-Yag laser (355 nm) drives a two successive-photon oxidative process of the dye. The first photon excites the dye to its first electronic singlet state. The second photon interacts with the excited molecule, ejects an electron to the solution and deactivates the molecule to a ground state of the oxidized dye (a+.). The oxidized product, a+., is an intensely colored compound ( A , , , = 445 nm, E = 43000 2 1000 M-I ern-') that reacts with a variety of electron donors like quinols, ascorbate and ferrous compounds. In the absence of added reductant, a+. is stable, having a lifetime of -10 min. In acidic solutions the solvated electrons generated by the photochemical reaction react preferentially with H+. In alkaline solution the favored electron acceptor is the ground-state pyranine anion and a radical, @-., of the reduced dye is formed. The reduced product is well distinguished from the oxidized one, having its maximal absorption at 510 nm with E = 25000 2 2000 M-l cm-l. The oxidized radical can be reduced either by @-. or by other electron donors. The apparent second-order rate constants of these reactions, which vary from lo6 u p to lo9 M-I s-l, are slower than the rates of diffusion-controlled reactions. Thus the redox reactions are limited by an energy barrier for electron transfer within the encounter complex between the reactants.

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Kinetics of flavin semiquinone reduction of the components of the cytochrome c-cytochrome b5 complex

Cited by 14 publications

References 26 publications

Laser flash photolysis studies of electron transfer to the cytochrome b5-cytochrome c complex

Laser flash photolysis studies of electron transfer to the cytochrome b5-cytochrome c complex

Genetic engineering of redox donor sites: measurement of intracomplex electron transfer between ruthenium-65-cytochrome b5 and cytochrome c

Fast Redox Perturbation of Aqueous Solution by Photoexcitation of Pyranine

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