EFFECT OF REPLACEMENT OF FERRIPROTOPORPHYRIN-IX IN THE HEME DOMAIN OFCYTOCHROME-P-450 BM-3 ON SUBSTRATE-BINDING AND CATALYTIC ACTIVITY

Bacillus megaterium cytochrome P-450 BM-3 (coded by gene CYP102) is a catalytically self-sufficient mono-oxygenase, with both cytochrome P-4 50 and NADPH:cytochrome P-450 reductase domains, that catalyses the hy droxylation of fatty acids. The natural ferriprotoporphyrin IX has bee n removed from the haem domain of cytochrome P-450 BM-3 by treatment w ith acidified acetone, and it has been shown that, under carefully con trolled conditions, haem can be added back to the resultant apoprotein to obtain a fully reconstituted haem domain with spectroscopic, subst rate-binding and catalytic properties indistinguishable from those of the native domain. Replacement of the natural haem with ferriprotoporp hyrin IX dimethyl ester yields a protein which has a higher affinity f or the substrate dodecanoic acid and (in the presence of the reductase domain) the same catalytic rate as the native haem domain. Replacemen t with ferrimesoporphyrin IX yields a protein with the same affinity f or 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 ha em iron effects the catalytic rate.