Pivotal Role of Water in the Mechanism of P450BM-3

Abstract

Cytochrome P450s constitute a superfamily of enzymes that catalyze the oxidation of a vast number of structurally and chemically diverse hydrophobic substrates. Herein, we describe the crystal structure of a complex between the bacterial P450BM-3 and the novel substrate N-palmitoylglycine at a resolution of 1.65 A, which reveals previously unrecognizable features of active site reorganization upon substrate binding. N-palmitoylglycine binds with higher affinity than any other known substrate and reacts with a higher turnover number than palmitic acid but with unaltered regiospecificity along the fatty acid moiety. Substrate binding induces conformational changes in distinct regions of the enzyme including part of the I-helix adjacent to the active site. These changes cause the displacement by about 1 A of the pivotal water molecule that ligands the heme iron, resulting in the low-spin to high-spin conversion of the iron. The water molecule is trapped close to the heme group, which allows it to partition between the iron and the new binding site. This partitioning explains the existence of a high-spin-low-spin equilibrium after substrate binding. The close proximity of the water molecule to the heme iron indicates that it may also participate in the proton-transfer cascade that leads to heterolytic bond scission of oxygen in P450BM-3.

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.…”

“…movement of the iron that directly impacts on the iron-ligand interactions, or movement of the palmitate molecule within the active site that enables a water molecule to ligate to, or dissociate from, the heme iron. A hydrophobic patch close to the mouth of the substrate access channel has been speculated to be the initial recognition site for fatty acids before the aliphatic chain enters the channel (Hasemann et al, 1995;Li and Poulos, 1997;Haines et al, 2001). If this patch remains accessible after the first fatty acid is bound, it may be an allosteric binding site.…”

Subtle structural changes in the Asp251Gly/Gln307His P450 BM3 mutant responsible for new activity toward diclofenac, tolbutamide and ibuprofen

“…Data were processed using XDS [27] and scaled using AIMLESS [28]. The structure was solved by molecular replacement using Molrep [29] from the CCP4 suite [30] and the coordinates of the heme domain of P450 BM3 structure (PDB 1JPZ) [22]. The models were adjusted using Coot [31] and refined using Refmac5 [32] and PHENIX [33].…”

“…The crystal structure of A2 mutant in complex with N-palmytoilglycine (NPG), a known substrate of P450 BM3 [22], was also solved and the electron density of the substrate was visible in one of the monomer ( Figure 4A). The overall structure is very similar to that of substrate-free A2 mutant (RMSD 0.583 Å).…”

“…Here, we report the crystal structure of the heme domain of the double mutant D251G/Q307H of cytochrome P450 BM3, named A2, previously generated and characterized from a catalytic point of view for its ability to generate metabolites of diclofenac, ibuprofen and tolbutamide [9]. The information from the crystal structure of the mutant also in complex with the substrate N-palmytoilglycine together with the comparison with the available structures of the wild type enzyme [21,22] were then used for a more detailed and comparative functional characterization, giving the rational basis to understand why the A2 mutant has evolved toward the recognition and the turnover of new substrates due to two mutations far from its active site.…”