Genetic variants in the putidaredoxin-cytochrome P-450cam electron-transfer complex: identification of the residue responsible for redox-state-dependent conformers.
Camphor is hydroxylated in Pseudomonas putida by a three-component system comprised of an oxidase, cytochrome P-450cam, and a two-protein electron-transfer chain, putidaredoxin and putidaredoxin reductase [Tyson et al. (1972) J. Biol. Chem. 274, 5777-5784]. The enzymatic removal of putidaredoxin's C-terminal tryptophan is known to cause a much reduced rate of enzymatic activity in the reconstituted camphor hydroxylase system [Sligar et al. (1974) Proc. Natl. Acad. Sci. U.S.A. 71, 3906-3910]. To further study the role of tryptophan in the association and/or electron-transfer reactions of putidaredoxin, the gene coding for the iron-sulfur protein was altered so that the tryptophan codon was either deleted or replaced by Phe, Tyr, Asp, Leu, Val, or Lys. Although the initial evaluation of these variant proteins [Davies et al. (1990) J. Am. Chem. Soc. 112, 7396-7398] showed much reduced velocities of electron transfer between P-450cam and the nonaromatic C-terminal proteins, the relative contributions of the binding specificity and intracomplex electron-transfer rates were not addressed. We report here a complete kinetic characterization of these proteins where the dependence of the rate constant on the putidaredoxin concentration was used to determine the intracomplex electron-transfer rate constants and the association energies for all the putidaredoxins in both oxidation states. The sum of forward and reverse intracomplex electron-transfer rate constants varies from 4.90 s-1 for the Lys C-terminal variant to 172 s-1 for the native protein.(ABSTRACT TRUNCATED AT 250 WORDS)
Davies
M D
MD
Department of Biochemistry, University of Illinois, Urbana, Champaign 61801.
Sligar
S G
SG
D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics*; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000235:genetics*; D004579:Electron Transport; D005288:Ferredoxins / Q000737:chemistry / Q000235:genetics*; D007700:Kinetics; D010084:Oxidation-Reduction
D001426:Bacterial Proteins; D005288:Ferredoxins; C100137:putidaredoxin; D003577:Cytochrome P-450 Enzyme System