NMR Studies of Substrate Binding to Cytochrome P450 BM3: Comparisons to Cytochrome P450 cam

Abstract

The binding of the substrates sodium laurate and sodium 12-bromolaurate to the heme-containing domain of Bacillus megaterium cytochrome P450 BM3 (CYP102) has been studied by measurement of the relaxation effects of the unpaired electrons of the heme iron on the protons of water 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 changes in the optical spectrum. The relaxation measurements show that this is accompanied by expulsion of water from the sixth coordination position of the iron, the distance between the iron and the water protons increasing from 2.6 to 5.2 A. Corresponding relaxation measurements 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 substrate in the binding site. Laurate and 12-bromolaurate are found to bind in 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 A from the heme iron. It is shown that laurate and pyridine can bind simultaneously to the P450 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 P450 cam, and their implications for structural changes involved in the catalytic cycle are discussed.

Inhibitor/fatty acid interactions with cytochrome P‐450 BM3

“…By contrast, SERRS indicates that binding of palmitate (and other fatty acids) to the haem domain causes enhancement of modes such as v29 (which is sensitive to the nature of the interaction of the haem ring with the immediate protein environment) but does not indicate direct interaction with the iron (no changes in v3 or v4). This finding is in agreement with the NMR data of Modi et al [23] and suggests an allosteric mechanism for v29 intensity increase. It would appear that, despite its distance, the binding of fatty acid is 'sensed' by the haem group of the enzyme, most likely through small deformations in the amino acid environment of the fatty acid being transmitted through to the haem.…”

“…Modi et al [23], used NMR to show that lauric acid was bound at nearly 0.8 nm from iron in oxidised P-450 BM3, sufficiently far to permit the co-binding of pyridine. This situation is different from P-450cam, where camphor/pyridine co-binding is not feasible.…”

Roles of key active-site residues in flavocytochrome P450 BM3

“…Previous studies have shown that laurate binding to wild-type P450 BM3 induces a low-to high-spin state shift of 30 % at saturating concentrations of laurate [21,32], that binding is characterized by a K d of approx. 90-110 µM (e.g.…”

Engineering the substrate specificity of Bacillus megaterium cytochrome P-450 BM3: hydroxylation of alkyl trimethylammonium compounds

“…Intact P-450 BM3 and its haem domain were expressed and purified to homogeneity as described previously [13,16]. The wild-type proteins were expressed in E. coli XL Blue 1 [13,16], and the mutant proteins in E. coli TG1.…”

“…Fatty-acid substrates initially bind relatively far from the haem, the terminal methyl being approx. 7.5 A H from the haem iron [13,14]. On reduction of the enzymesubstrate complex there is a conformational change which leads to a 6 A H movement of the substrate towards the haem, into…”