TY - JOUR
T1 - Genetic Variants in the Putidaredoxin-Cytochrome P-450cam Electron-Transfer Complex
T2 - Identification of the Residue Responsible for Redox-State-Dependent Conformers
AU - Davies, Matthew D.
AU - Sligar, Stephen G.
PY - 1992/2/1
Y1 - 1992/2/1
N2 - 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-450oam 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−1for the Lys C-terminal variant to 172 s−1for the native protein. Differences in the behavior of the variant proteins are most striking when comparing the cytochrome P-450cam association energies with reduced putidaredoxins. The presence of a C-terminal aromatic residue is required for a relatively high cytochrome P-450cam affinity of the reduced relative to the oxidized protein. The desolvation of putidaredoxin's C-terminal residue is discussed as a possible explanation for this behavior.
AB - 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-450oam 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−1for the Lys C-terminal variant to 172 s−1for the native protein. Differences in the behavior of the variant proteins are most striking when comparing the cytochrome P-450cam association energies with reduced putidaredoxins. The presence of a C-terminal aromatic residue is required for a relatively high cytochrome P-450cam affinity of the reduced relative to the oxidized protein. The desolvation of putidaredoxin's C-terminal residue is discussed as a possible explanation for this behavior.
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U2 - 10.1021/bi00161a016
DO - 10.1021/bi00161a016
M3 - Article
C2 - 1445875
AN - SCOPUS:0026442895
SN - 0006-2960
VL - 31
SP - 11383
EP - 11389
JO - Biochemistry
JF - Biochemistry
IS - 46
ER -