The binding domain on horse cytochrome c and Rhodobacter sphaeroides cytochrome c2 for the Rhodobacter sphaeroides cytochrome bc1 complex

Abstract

The interaction of the Rhodobacter sphaeroides cytochrome bc1 complex with Rb. sphaeroides cytochrome c2 and horse cytochrome c was studied by using specific lysine modification and ionic strength dependence methods. The rate of the reactions with both cytochrome c and cytochrome c2 decreased rapidly with increasing ionic strength above 0.2 M NaCl. The ionic strength dependence suggested that electrostatic interactions were equally important to the reactions of the two cytochromes, even though they have opposite net charges at pH 7.0. In order to define the interaction domain on horse cytochrome c, the reaction rates of derivatives modified at single lysine amino groups with trifluoroacetyl or trifluoromethylphenylcarbamoyl were measured. Modification of lysine-8, -13, -27, -72, -79, and -87 surrounding the heme crevice was found to significantly lower the rate of the reaction, while modification of lysines in other regions had no effect. This result indicates that lysines surrounding the heme crevice of horse cytochrome c are involved in electrostatic interactions with carboxylate groups at the binding site on the cytochrome bc1 complex. In order to define the reaction domain on cytochrome c2, a fraction consisting of a mixture of singly labeled 4-carboxy-2,6-dinitrophenylcytochrome c2 derivatives modified at lysine-35, -88, -95, -97, and -105 and several unidentified lysines was prepared. Although it was not possible to resolve these derivatives, all of the identified lysines are located on the front surface of cytochrome c2 near the heme crevice. The rate of reaction of this fraction was significantly smaller than that of native cytochrome c2, suggesting that the binding domain on cytochrome c2 is also located at the heme crevice.(ABSTRACT TRUNCATED AT 250 WORDS)

Demonstration of Short-lived Complexes of Cytochrome c with Cytochrome bc1 by EPR Spectroscopy

“…5 (43); the second method was a nonlinear fitting of a biexponential function to the SR curves (see “Experimental Procedures” for details). Because the binding between the two redox partners is in part controlled by electrostatic interactions, (23–27, 56, 57) the changes in the ionic strength were used to effectively manipulate the binding equilibrium of the reaction.…”

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

“…To compare the interaction of Ru-72-Cc and native horse cytochrome c with R. sphaeroides cytochrome bc 1 , steady-state kinetics were carried out under high ionic strength conditions, where previous studies have shown that the Michaelis constant K m is a good measure of the binding interaction (22). Native horse cytochrome c was found to have a V max of 3.8 Ϯ 0.5 M cytochrome c reduced per min/nM cytochrome b and a K m of 10 Ϯ 1 M in buffer containing 25 mM sodium phosphate (pH 7.0), 150 mM NaCl, and 0.3 mM EDTA.…”

“…The rate of nonenzymatic reduction of cytochrome c by Q 0 C 10 BrH 2 , determined under the same conditions in the absence of enzyme, was subtracted. The steady-state parameters V max and K m were determined from EadieHofstee plots of the initial velocities as described (22).…”

Respiratory electron transport and light-induced energy transduction in membranes from the aerobic photosynthetic bacterium Roseobacter denitrificans

“…These data, when examined with the integrated Michaelis-Menten equation, yielded a K m in the order of 0.4 µM which is similar to that reported for the reduction of Rba. sphaeroides cytochrome c 2 by mitochrondrial bc 1 complex (Hall et al 1987). Further, the rate of oxygen consumption catalyzed by membrane fragments (30 nM bc 1 complex) with Na-ascorbate plus cytochrome c 551 as donors was determined to test the efficiency of cytochrome c 551 in reducing the terminal oxidase.…”