Genetic Variants in the Putidaredoxin-Cytochrome P-450(cam) Electron Transfer Complex: Identification of the Residue Responsible for Redox-State Dependent Complex Stabilization
Davies, Matthew Donald
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https://hdl.handle.net/2142/72351
Description
Title
Genetic Variants in the Putidaredoxin-Cytochrome P-450(cam) Electron Transfer Complex: Identification of the Residue Responsible for Redox-State Dependent Complex Stabilization
Author(s)
Davies, Matthew Donald
Issue Date
1992
Doctoral Committee Chair(s)
Sligar, Stephen G.
Department of Study
Biochemistry
Discipline
Biochemistry
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Chemistry, Biochemistry
Abstract
Camphor is hydroxylated in Pseudomonas putida by a three component system comprised of an oxidase, cytochrome P-450$\sb{\rm cam},$ 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. USA 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. The reaction rates for the camphor hydroxylase system reconstituted with native C-terminal tryptophan or aromatic substituted putidaredoxins are at least ten-fold higher than the average rates for reconstitutions with non-aromatic substituted proteins. The reduction of the oxy-cytochrome P-450$\sb{\rm cam}$ by Pd is the rate limiting step in the electron transfer chain, and the stoichiometry of NADH to product depends on the concentration of non-aromatic variant putidaredoxins. In an attempt to more completely characterize the electron transfer behavior of the variant proteins, experiments were undertaken to separate the "first" reduction of P-450$\sb{\rm cam}$ from later catalytic steps. The dependence of the observed rate constant on the putidaredoxin concentration was used to determine the intra-complex electron transfer rate constants and the association energies for all the putidaredoxins in both oxidation states. The sums of forward and reverse intra-complex electron transfer rate constants vary from 4.90 s$\sp{-1}$ for the Lys C-terminal variant to 172 s$\sp{-1}$ for the native protein. Differences in the behavior of the variant proteins are greatest when comparing the cytochrome P-450$\sb{\rm cam}$ association energies with reduced putidaredoxins. The presence of a C-terminal aromatic residue is required for a relatively high cytochrome P-450$\sb{\rm cam}$ affinity (average difference = 78 meV) 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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