Effect of replacement of ferriprotoporphyrin IX in the haem domain of cytochrome P-450 BM-3 on substrate binding and catalytic activity

Abstract

Bacillus megaterium cytochrome P-450 BM-3 (coded by gene CYP102) is a catalytically self-sufficient mono-oxygenase, with both cytochrome P-450 and NADPH:cytochrome P-450 reductase domains, that catalyses the hydroxylation of fatty acids. The natural ferriprotoporphyrin IX has been removed from the haem domain of cytochrome P-450 BM-3 by treatment with acidified acetone, and it has been shown that, under carefully controlled conditions, haem can be added back to the resultant apoprotein to obtain a fully reconstituted haem domain with spectroscopic, substrate-binding and catalytic properties indistinguishable from those of the native domain. Replacement of the natural haem with ferriprotoporphyrin IX dimethyl ester yields a protein which has a higher affinity for the substrate dodecanoic acid and (in the presence of the reductase domain) the same catalytic rate as the native haem domain. Replacement with ferrimesoporphyrin IX yields a protein with the same affinity for substrate, but a reduced catalytic turnover. These results suggest that the haem moiety has a role in the creation of the binding pocket for substrate, and that modification of the electron density on the haem iron effects the catalytic rate.