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

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25 Citations (Scopus)


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 (Kd ∇ 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 (Kd ∇ 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 Kd, 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 m V 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 oxygenactivating P450s) the evolution of XplA as a specialized RDX reductase catalyst.

Original languageEnglish
Pages (from-to)19699-19714
Number of pages16
JournalJournal of Biological Chemistry
Issue number23
Early online date12 Apr 2012
Publication statusPublished - 1 Jun 2012
Externally publishedYes


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