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

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.

Name thesis:degree_name

Ph.D.

Level thesis:degree_level

Dissertation

Discipline thesis:degree_discipline

Physics

Year dc:date

2012

Author dc:creator

• Sligar, Stephen Gary

Contributors dc:contributor

• Debrunner, Peter G.
• Gunsalus, I.C.

• camphor
• Biochemistry
• physics
• catabolic pathway
• enzymes
• Pseudomonas putida

Statement dc:rights

• © 1975 Stephen Gary Sligar

Language dc:language

en

Identifier

2230997

OAI identifier oai:identifier

oai:www.ideals.illinois.edu:2142/30763