Laser flash photolysis studies of electron transfer to the cytochrome b5-cytochrome c complex.
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)
Department of Biochemistry, University of Arizona, Tucson 85721.
T E
TE
Meyer
M
M
Rivera
F A
FA
Walker
M R
MR
Mauk
A G
AG
Mauk
M A
MA
Cusanovich
G
G
Tollin
D000818:Animals; D002417:Cattle; D003001:Cloning, Molecular; D003574:Cytochrome c Group / Q000737:chemistry*; D015786:Cytochromes b5 / Q000737:chemistry*; D004579:Electron Transport; D004926:Escherichia coli; D006736:Horses; D007834:Lasers; D008862:Microsomes, Liver / Q000201:enzymology; D008928:Mitochondria / Q000201:enzymology; D009994:Osmolar Concentration; D010782:Photolysis; D051381:Rats
D003574:Cytochrome c Group; D015786:Cytochromes b5