Genetic engineering of redox donor sites: measurement of intracomplex electron transfer between ruthenium-65-cytochrome b5 and cytochrome c.
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)
Willie
A
A
Department of Chemistry and Biochemistry, University of Arkansas, Fayetteville 72701.
Stayton
P S
PS
Sligar
S G
SG
Durham
B
B
Millett
F
F
D000595:Amino Acid Sequence; D001665:Binding Sites; D003545:Cysteine; D003574:Cytochrome c Group / Q000737:chemistry / Q000378:metabolism*; D015786:Cytochromes b5 / Q000737:chemistry / Q000378:metabolism*; D004579:Electron Transport; D007700:Kinetics; D007834:Lasers; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010446:Peptide Fragments / Q000302:isolation & purification; D010782:Photolysis; D011487:Protein Conformation; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012428:Ruthenium
D003574:Cytochrome c Group; D010446:Peptide Fragments; D011994:Recombinant Proteins; D012428:Ruthenium; D015786:Cytochromes b5; D003545:Cysteine