NMR-STUDIES OF SUBSTRATE-BINDING TO CYTOCHROME-P-450 (BM3) - COMPARISONS TO CYTOCHROME-P-450 (CAM)

The binding of the substrates sodium laurate and sodium 12-bromolaurat e to the heme-containing domain of Bacillus megaterium cytochrome P-45 0 (BM3) (CYP102) has been studied by measurement of the relaxation eff ects of the unpaired electrons of the heme iron on the protons of wate r and of the bound substrates. Substrate binding leads to a conversion of the heme iron from a low-spin to a high-spin state, as shown by ch anges in the optical spectrum. The relaxation measurements show that t his is accompanied by expulsion of water from the sixth coordination p osition of the iron, the distance between the iron and the water proto ns increasing from 2.6 to 5.2 Angstrom. Corresponding relaxation measu rements on the substrate protons lead to the determination of a number of distances between the iron and protons of the bound substrate and, hence, to information on the position and orientation of the substrat e in the binding site. Laurate and 12-bromolaurate are found to bind i n a very similar way, in an extended conformation with the carboxylate probably close to Arg47 and the other end of the chain 7.6-7.8 Angstr om from the heme iron. It is shown that laurate and pyridine can bind simultaneously to the P-450 domain and that the iron-laurate distances in this ternary complex are not significantly different from those in the binary complex. These observations are compared with those on the substrate complex of cytochrome P-450 (cam), and their implications f or structural changes involved in the catalytic cycle are discussed.