Cytochrome P450, a very hard mountain to climb: evidence for multiple functional species of activated oxygen

Although much has been learned about cytochrome P450 as an oxygenating catalyst since its discovery as a carbon monoxide-binding pigment in liver microsomes over 40 years ago, two major problems remain unsolved. These are the role of the heme sulfur ligand and the identity of the “activated oxygen” species generated during the reduction of molecular oxygen. To address these questions, mutagenesis of pertinent amino acid residues in NH2-terminal-truncated microsomal P450s 2B4 and 2E1, has been carried out. Clones for these cytochromes with the active-site cysteine replaced by histidine, serine, or tyrosine were expressed in Escherichia coli. Despite instability, some of the resulting hemoproteins were purified. The histidine-437 mutant of P450 2E1 is reduced by NADPH in the presence of the reductase and yields H2O2 in the reconstituted system. However, no activity was detected in the oxidation of several substrates, thus indicating a functional role for the sulfur ligand. In other experiments, the active-site threonine residue that facilitates proton transfer was replaced with alanine. Changes in the rates of catalysis of aldehyde deformylation, olefin epoxidation, ipso-substitution, and other reactions by P450 2B4:T302A and 2E1:T303A mutants provided evidence for peroxo-iron, hydroperoxo-iron, and oxenoid-iron as discrete functional oxygenating species. The availability of multiple oxidants is believed to contribute to the unmatched versatility of the P450 cytochromes.