This review discusses the mechanisms of oxygen activation by cytochrome P450 enzymes, the possible catalytic roles of the various iron—oxygen species formed in the catalytic cycle, and progress in understanding the mechanisms of hydrocarbon hydroxylation, heteroatom oxidation, and olefin epoxidation. The focus of the review is on recent results, but earlier work is discussed as appropriate. The literature through to February 2002 is surveyed, and 175 referenced are cited. Covering: up to February 2002.

Bovine endothelial nitric-oxide synthase (eNOS) expressed in Escherichia coli does not have the post-translational modifications found in the native enzyme and is free of tetrahydrobiopterin (BH). In the presence of BH, eNOS has an absorption maximum at 400 nm that shifts to 395 nm when the substrate L-arginine is added. The low-spin component of the spectrum of the BH-free protein is decreased by the addition of BH without a corresponding increase in the high-spin component. Addition of BH decreases the low-spin population of eNOS even in the presence of excess L-arginine. These results indicate that BH directly modulates the heme environment. BH-free eNOS is completely inactive, but catalytic activity is recovered when BH (EC 200 nM) is added. The spectroscopically determined binding constants for L-arginine are 1.9 μM in the presence and 4.0 μM in the absence of BH. The BH-supplemented enzyme has an activity of 90-120 nmol of citrulline•min•mg and K values of 3 and 14 μM for L-arginine and N-hydroxy-L-arginine, respectively. Of particular interest is the finding by SDS-polyacrylamide gel electrophoresis that BH-free eNOS exists in a monomer-dimer equilibrium very similar to that observed with the BH-reconstituted protein. Addition of BH increases the percent of the dimer by only 5%. The results establish that BH influences the heme environment and stabilizes the protein with respect to heme loss but is not required for dimer formation.