Evaluation of the Functional Role of the Heme-6-propionate Side Chain in Cytochrome P450cam

Cytochrome P450cam (P450cam) binds a protoheme IX as a prosthetic group via noncovalent interactions. Heme-6-propionate, one of the two heme-propionate side chains, forms hydrogen-bonding interactions with Arg112 and other hydrophilic amino acid residues. Here, we demonstrate the structural and functional roles of the 6-propionate side chain in P450cam using a reconstituted protein with 6-depropionate-6-methylated protoheme IX (one-legged heme). The spectroscopic data and the enzymatic activities reveal that removal of the 6-propionate has no clear influence on the enzyme property. The rate of electron transfer from putidaredoxin (Pdx), a natural redox partner, to P450cam was not significantly changed, whereas, the removal of the 6-propionate decreased the affinity of Pdx by 3.5-fold supporting the proposed role of Arg112 as the essential constituent of the Pdx binding site. Resonance Raman experiments indicate that removal of the 6-propionate weakens the Fe−S bond strength. The X-ray structure of the reconstituted protein at 1.55 Å resolution is highly superimposable with that of the wild-type protein, whereas the thiolate of the Cys357 heme ligand in the reconstituted protein is visible from the protein surface owing to the lack of the 6-propionate. Lengthening of the Fe−S bond and the water accessibility could facilitate protonation of thiolate anion to thiol, explaining the observed formation of the inactive P420 species under the mild conditions. Therefore, the d -camphor hydroxylation reaction requires a 6-propionate−protein matrix interaction to maintain an active P450 species.