Engineering cytochrome c peroxidase into cytochrome P450: A proximal effect on heme-thiolate ligation

In an effort to investigate factors required to stabilize heme-thiolate lig ation, key structural components necessary to convert cytochrome c peroxida se (CcP) into a thiolate-ligated cytochrome P450-like enzyme have been eval uated and the H175C/D235L CcP double mutant has been engineered. The UV-vis ible absorption, magnetic circular dichroism (MCD) and electron paramagneti c resonance (EPR) spectra for the double mutant at pH 8.0 are reported here in. The close similarity between the spectra of ferric substrate-bound cyto chrome P350cam and those of the exogenous ligand-free ferric state of the d ouble mutant with all three techniques support the conclusion that the latt er has a pentacoordinate, high-spin heme with thiolate ligation. Previous e fforts to prepare a thiolate-ligated mutant of CcP with the H175C single mu tant led to Cys oxidation to cysteic acid [Choudhury ct al. (1994) J. Biol, Chem. 267, 25656-25659]. Therefore it is concluded that changing the proxi mal Asp235 residue to Leu is critical in forming a stable heme-thiolate lig ation in the resting state of the enzyme. To further probe the versatility of the CcP double mutant as a ferric P450 model, hexacoordinate low-spin co mplexes have also been prepared, Addition of the neutral ligand imidazole o r of the anionic ligand cyanide results in formation of hexacoordinate addu cts that retain thiolate ligation as determined by spectral comparison to t he analogous derivatives of ferric P450cam. The stability of these complexe s and their similarity to the analogous forms of P450cam illustrates the po tential of the H175C/D235L CcP double mutant as a model for ferric P450 enz ymes. This study marks the first time a stable cyanoferric complex of a mod el P450 has been made and demonstrates the importance of the environment ar ound the primary coordination ligands in stabilizing metal-ligand ligation.