HYDROXYBENZOYL-COA REDUCTASE - COUPLING KINETICS AND ELECTROCHEMISTRYTO DERIVE ENZYME MECHANISMS

Hydroxybenzoyl-CoA reductase (HBCR) is an iron-sulfur protein that is involved in the metabolism of aromatic compounds. It catalyzes the two -electron reduction of hydroxybenzoyl-CoA to benzoyl-CoA. In the work described here, kinetic schemes were derived for HBCR and for several classes of redox enzymes and redox-activated enzymes. Introduction of the Nernst equation into the rate laws led to the development of novel relationships between the ambient redox potential, the midpoint poten tial of the enzyme active site, and the kinetic parameter, V/K. By cou pling electrochemistry and steady-state kinetics, mechanistic informat ion could be obtained that could not be determined by either method al one. For HBCR, the relationship between the kinetic parameter V/K and the ambient electrochemical potential of the assay mixture was found t o be: apparent V/K-m = V-max/{K-m{1 + exp[nF/RT(E - E degrees(e))]}}, where n is the number of electrons involved in the redox process, F is the Faraday constant, R is the gas constant, T is the temperature in K, E is the applied potential, and E degrees(e) is the redox potential of a redox-active catalytic site on the enzyme. Coupling kinetics wit h electrochemistry yielded the E degrees(e) (-350 mV vs NHE) for HBCR and maximum values under optimal redox conditions for k(cat) and k(cat )/K-m (9 s(-1) and 1.8 x 10(5) M(-1) s(-1), respectively). In addition , theory was developed that could distinguish a single two-electron tr ansfer mechanism from one involving two successive one-electron transf ers. HBCR was found to be in the latter class. Interestingly, the deri ved mechanism for HBCR is similar to that of the Birch reduction, the classical organic chemical reaction for reductive dehydroxylation of p henolic compounds. The methodology described here represents a novel a pproach that should help elucidate the mechanisms of other oxidoreduct ase and redox-activated enzymes.