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

Abstract

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)

Definition of the Interaction Domain for Cytochrome con the Cytochrome bc 1 Complex

“…The rate constant for intracomplex electron transfer with R. sphaeroides cytochrome bc 1 is also nearly independent of ionic strength. Similar behavior has also observed in the reactions of cytochrome c with purified cytochrome c 1 (17), cytochrome oxidase (16,27), cytochrome c peroxidase (28 -31), and cytochrome b 5 (32,33).…”

Definition of the Interaction Domain for Cytochrome con Cytochrome c Oxidase

“…This indicates that the orientation of the 1:1 complex is optimal for electron transfer at low ionic strength and does not change as the ionic strength is increased. Similar behavior is also observed in the reactions of Cc with beef CcO (19,20), cytochrome c 1 (39), cytochrome c peroxidase (40 -44), and cytochrome b 5 (45,46). Experiments using soluble reducing agents led to the suggestion that the Cc⅐CcO complex may not be optimized for rapid electron transfer at low ionic strength (47).…”

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

“…In such cases, one would expect a monotonic decrease in rate constant with ionic strength, such as has been observed by Willie et al (1992) for cytochrome b,-cytochrome c.…”

“…The interaction of cytochrome b5 with cytochrome c has been computer-modeled by Salemme (1976), by Mauk et al (1986), and by Wendoloski et al (1987), and an interaction energy for various orientations of -5.7 to -6.7 kcal/mol at 4 mM ionic strength was calculated (Mauk et al, 1986). Rate constants versus ionic strength were measured for the cytochrome b5-cytochrome c electron transfer system by Willie et al (1992), and we have calculated the magnitude of the electrostatic interaction using Equation 5 with a single parameter ( vj = -11.9). The interaction energy at I = 4 mM was obtained from the fitting parameter Yi using Equation 5d and the relation VToT = K j X ( I ) k T (kT is 0.59 at 25 "C).…”