Cytochrome P450cam (P450cam) binds a protoheme IX as a prosthetic group via noncovelent interactions. Heme-6-propionate, one of the two hems-propionate side chains. forms hydrogen-banding interactions with Arg112 and other hydrophilic amino acid residues. Here, we demonstrate the structural and functional rates of the 6-propionate side chain in P450cam using a reconstituted protein with 6- depropionate 6-methylated protoheme IX (one-legged hems). 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 radox partner, to P450cam was not significantly changed. whereas, the removd 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 Roman experiments indicate that removal of the 6-propionate weakens the Fe-S bond strength. The Xray structure of the reconstituted protein at 1.55A resolution is highly superimposable with that of the wild-type protein, whereas the thiolate of the Cys357 hems 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 protonationof 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.