MOLECULAR RECOGNITION IN CYTOCHROME-P-450 - MECHANISM FOR THE CONTROL OF UNCOUPLING REACTIONS

The pathway for utilization of pyridine nucleotide derived reducing equivalents in the cytochrome P-450 monooxygenase systems has three major branch points. The first is a partitioning between autoxidation of a ferrous, oxygenated heme adduct and input of the second reducing equivalent required for monooxygenase stoichiometry. The second is between dioxygen bond scission and release of two-electron-reduced O2 as hydrogen peroxide. The third is between substrate hydrogen abstraction initiated by a putative higher valent iron-oxo species and reduction of this intermediate by two additional electrons to produce water in an overall oxidase stoichiometry. For all substrates investigated, the direct release of superoxide at the first branch point never competes with second electron input. In order to elucidate the aspects of molecular recognition of a substrate-P-450 complex which affect these individual branch points in the catalytic cycle, we have measured the NADH-derived reducing equivalents recovered in hydroxylated substrate, hydrogen peroxide, and water for a series of active-site mutants designed to alter the coupling of ethylbenzene hydroxylation. We find that the reaction specificity at the second and third branch points is affected by site-directed mutations that alter the topology of the binding pocket. The increased commitment to catalysis observed for all mutants suggests that active-site hydration is important in the uncoupling to form hydrogen peroxide at the second branch point. The liberation of hydrogen peroxide does not correlate with the location of the mutation in the pocket, as expected if the two-electron-reduced dioxygen-bound intermediate is not directly participating in the substrate activation step. However, a strong correlation is observed between water production at the third branch point and the location and size of the amino acid side chain in the substrate binding site. Larger hydrophobic side chains introduced in the upper regions of the binding pocket increase the ratio of hydroxylated product to water production by 2-4-fold relative to wild-type, while similar substitutions in residues near the heme plane result in diminished product. Overall, the partitioning between hydroxylation and oxidase activities varies by over 65% due to the location of nonpolar substituents engineered into the active site. Substrate access to the heme is the key factor for tight coupling at the level of the putative iron-oxo species. These results are further evidence that a discrete intermediate containing a single oxygen atom (e.g., a ferryl-oxo complex, [FeO]3+) is the precursor to both substrate hydroxylation and the input of two additional electron equivalents to form water.