Unusual Spectroscopic and Ligand Binding Properties of the Cytochrome P450-Flavodoxin Fusion Enzyme XplA

Soi H Bui,Kirsty J Mclean,Myles R Cheesman,Justin M Bradley,Stephen E.J Rigby,Colin W Levy,David Leys,Andrew W Munro

doi:10.1074/jbc.m111.319202

Soi H Bui, Kirsty J Mclean + Show 6 more

Open Access

https://doi.org/10.1074/jbc.m111.319202

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Abstract

The Rhodococcus rhodochrous strain 11Y XplA enzyme is an unusual cytochrome P450-flavodoxin fusion enzyme that catalyzes reductive denitration of the explosive hexahydro-1,3,5-trinitro-1,3,5-triazene (RDX). We show by light scattering that XplA is a monomeric enzyme. XplA has high affinity for imidazole (K(d) = 1.6 μM), explaining previous reports of a red-shifted XplA Soret band in pure enzyme. The true Soret maximum of XplA is at 417 nm. Similarly, unusually weak XplA flavodoxin FMN binding (K(d) = 1.09 μM) necessitates its purification in the presence of the cofactor to produce hallmark flavin contributions absent in previously reported spectra. Structural and ligand-binding data reveal a constricted active site able to accommodate RDX and small inhibitory ligands (e.g. 4-phenylimidazole and morpholine) while discriminating against larger azole drugs. The crystal structure also identifies a high affinity imidazole binding site, consistent with its low K(d), and shows active site penetration by PEG, perhaps indicative of an evolutionary lipid-metabolizing function for XplA. EPR studies indicate heterogeneity in binding mode for RDX and other ligands. The substrate analog trinitrobenzene does not induce a substrate-like type I optical shift but creates a unique low spin EPR spectrum due to influence on structure around the distal water heme ligand. The substrate-free heme iron potential (-268 mV versus NHE) is positive for a low spin P450, and the elevated potential of the FMN semiquinone/hydroquinone couple (-172 mV) is also an adaptation that may reflect (along with the absence of a key Thr/Ser residue conserved in oxygen-activating P450s) the evolution of XplA as a specialized RDX reductase catalyst.

Highlights

XplA is a P450-flavodoxin fusion catalyzing reduction of the explosive RDX
XplA is an unusual member of the P450 superfamily, which has become specialized to catalyze reductive denitration as an initial step in the degradation and detoxification of the military explosive RDX [21]
It is an example of a growing class of P450 enzymes fused to redox partners or other protein modules, with a flavodoxin domain attached at the N-terminal of the XplA P450 [15, 22]

Summary

Background

XplA is a P450-flavodoxin fusion catalyzing reduction of the explosive RDX. Results: XplA has unusual properties of high P450 affinity for imidazole and weak flavodoxin FMN binding. Studies using 18O2 and H218O demonstrated that a labeled oxygen was incorporated into 4-nitro-2,4-diazabutanal product only from H218O, apparently ruling out redox partnerdependent P450 heme iron-mediated oxygen insertion chemistry from either CYP2B4 or the Rhodococcus (XplA) P450 [25] This conclusion is consistent with the observation that a phylogenetically conserved cytochrome P450 Ser/Thr residue that participates in oxygen activation is absent in XplA [1, 26]. We provide here a detailed study of the spectroscopic and thermodynamic properties of XplA These reveal several peculiar features, including unusually weak binding of FMN to the XplA flavodoxin module, high affinity of the P450 heme for imidazole (and Tris) that bind weakly to most P450s, and a very positive heme iron reduction potential. Our data reveal several non-standard properties for a P450 system adapted as a reductive (rather than an oxidative) catalyst

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DISCUSSION