A Single Mutation in Cytochrome P450 BM3 Induces the Conformational Rearrangement Seen upon Substrate Binding in the Wild-type Enzyme

Abstract

The multidomain fatty-acid hydroxylase flavocytochrome P450 BM3 has been studied as a paradigm model for eukaryotic microsomal P450 enzymes because of its homology to eukaryotic family 4 P450 enzymes and its use of a eukaryotic-like diflavin reductase redox partner. High-resolution crystal structures have led to the proposal that substrate-induced conformational changes lead to removal of water as the sixth ligand to the heme iron. Concomitant changes in the heme iron spin state and heme iron reduction potential help to trigger electron transfer from the reductase and to initiate catalysis. Surprisingly, the crystal structure of the substrate-free A264E heme domain mutant reveals the enzyme to be in the conformation observed for substrate-bound wild-type P450, but with the iron in the low-spin state. This provides strong evidence that the spin-state shift observed upon substrate binding in wildtype P450 BM3 not only is caused indirectly by structural changes in the protein, but is a direct consequence of the presence of the substrate itself, similar to what has been observed for P450cam. The crystal structure of the palmitoleate-bound A264E mutant reveals that substrate binding promotes heme ligation by Glu 264 , with little other difference from the palmitoleate-bound wildtype structure observable. Despite having a protein-derived sixth heme ligand in the substrate-bound form, the A264E mutant is catalytically active, providing further indication for structural rearrangement of the active site upon reduction of the heme iron, including displacement of the glutamate ligand to allow binding of dioxygen.

Flavocytochrome P450 BM3 Mutant A264E Undergoes Substrate-dependent Formation of a Novel Heme Iron Ligand Set

“…The shorter chain fatty acids, with their carboxylate still tethered at the active-site mouth, are predicted not to extend far enough toward the heme to enable interaction with the glutamate. Again, these predictions are supported by structural data (see accompanying article (72)). …”

“…Structural reasons underlying this substrate-induced equilibrium perturbation are described in the accompanying article (72). Differences in active-site architecture between P450 BM3 and the mammalian CYP4 enzymes likely explain the failure of the A264E P450 BM3 variant to covalently link the heme macrocycle to the glutamate side chain.…”

The Crystal Structure of Cytochrome P450 4B1 (CYP4B1) Monooxygenase Complexed with Octane Discloses Several Structural Adaptations for ω-Hydroxylation

“…Moreover, the formation of the ester bond creates a less polar active site for the binding of the hydrocarbon. In contrast, the charged non-esterified glutamate might stabilize the binding of water in the active site or interact directly with the heme iron to compete with substrate binding as seen in a structure of the P450 BM3 A264E mutant (48). As Glu-310 is tethered to the heme, no unfavorable loss of Glu-310 side-chain conformational entropy is associated with substrate binding.…”

Chain length-dependent cooperativity in fatty acid binding and oxidation by cytochrome P450BM3 (CYP102A1)

“…The mechanism by which four palmitate molecules could bind to each monomer of dimeric full-length P450 BM3 or the heme domain is not clear. Crystal structures of the heme domain complexed with a fatty acid or derivative show only one substrate molecule in the active site, and in all cases the heme iron is penta-coordinate and almost certainly high spin (Hasemann et al, 1995;Li and Poulos, 1997;Haines et al, 2001;Joyce et al, 2004;Hegde et al, 2007;Huang et al, 2007;Haines et al, 2008). However, limited fatty acid solubility, the high protein concentrations and ionic strengths required for crystallization, and crystal packing effects mean that multiple fatty acid binding may not be observable in crystal structures.…”