University of Illinois Urbana-Champaign

A kinetic and equilibrium description of camphor hydroxylation by the P450cam monoxygenase system

Sligar, Stephen Gary

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Description

Title
A kinetic and equilibrium description of camphor hydroxylation by the P450cam monoxygenase system
Author(s)
Sligar, Stephen Gary
Issue Date
1975
Director of Research (if dissertation) or Advisor (if thesis)
  • Debrunner, Peter G.
  • Gunsalus, I.C.
Department of Study
Physics
Discipline
Physics
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
  • camphor
  • Biochemistry
  • physics
  • catabolic pathway
  • enzymes
  • Pseudomonas putida
Language
en
Abstract
Camphor is hydroxylated in the soil bacterium Pseudomonas putida by a soluble three protein monoxygenase system consisting of (1) a NADH-specific FAD flavoprotein reductase, (2) a Fe2S*2 Cys4 iron-sulfur redoxin termed putidaredoxin (Pd) and (3) cytochrome P450cam, abbreviated cytochrome m for monoxygenase. The interaction of Pd and cytochrome m in the overall monoxygenase reaction is studied by equilibrium and kinetic techniques. Three compounds (termed effectors) are shown to react with oxygenated cytochrome m in the generation of product: Pdr , a modified putidaredoxin (Pdr.dTrp), and dihydrolipoic acid. In all cases a kinetic analysis of the product forming reaction indicates that an effector m02rs complex precedes camphor hydroxylation. Cytochrome m is shown by equilibrium methods to bind two molecules of putidaredoxin. Pd binding at one site induces a shift in the Soret absorption maximum of cytochrome m corresponding to an observed difference spectrum with peak at 420 nm and trough at 386 nm. The Pd-cytochrome m complex formed by ligation at this site is that indicated by kinetic methods to be the obligatory intermediate in the generation of product from m02rs. Pd binding at a second cytochrome m locus is observed by the quenching of fluorescence from a dye label attached to the external sulfhydryl group on cytochrome m. Regulation of oxidation/reduction potential is observed by ligation at this site and the data are presented in the form of a free energy diagram graphically indicating the coupling between redox and binding energies.
Type of Resource
text
Permalink
http://hdl.handle.net/2142/30763
Copyright and License Information
© 1975 Stephen Gary Sligar

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