Biophysical Characterization of the Sterol Demethylase P450 from Mycobacterium tuberculosis, Its Cognate Ferredoxin, and Their Interactions

Abstract

Mycobacterium tuberculosis encodes a P450 of the sterol demethylase family (CYP51) chromosomally located adjacent to a ferredoxin (Fdx). CYP51 and Fdx were purified to homogeneity and characterized. Spectroscopic analyses were consistent with cysteinate- and aqua-ligated heme iron in CYP51. An epsilon419 of 134 mM(-1) cm(-1) was determined for oxidized CYP51. Analysis of interactions of 1-, 2-, and 4-phenylimidazoles with CYP51 showed that the 1- and 4-forms were heme iron-coordinating inhibitors, while 2-phenylimidazole induced a substrate-like optical shift. The 2-phenyimidazole-bound CYP51 demonstrated unusual decreases in high-spin heme iron content at elevated temperatures and an almost complete absence of high-spin heme iron by low-temperature EPR. These data suggest thermally induced alterations in CYP51 active site structure and/or binding modes for the small ligand. Reduction of CYP51 in the presence of carbon monoxide leads to formation of an Fe(II)-CO complex with a Soret absorption maximum at 448.5 nm, which collapses (at 0.246 min(-1) at pH 7.0) forming a species with a Soret maximum at 421.5 nm (the inactive P420 form). The rate of P420 formation is accelerated at lower pH, consistent with protonation of the cysteinate (Cys 394) to a thiol underlying the P450-P420 transition. The P450 form is stabilized by estriol, which induces a type I spectral shift on binding CYP51 (Kd = 21.7 microM). Nonstandard spectral changes occur on CYP51 reduction (using either dithionite or natural redox partners), including a blue-shifted Soret band and development of a strong feature at approximately 558.5 nm, suggestive of cysteine thiol ligation. Thus, ligand-free ferrous CYP51 is prone to thiolate ligand protonation even in the absence of carbon monoxide. Analysis of reoxidized CYP51 demonstrates that the enzyme re-forms P450, indicating that Cys 394 thiol is readily deprotonated to thiolate in the ferric form. Spectroscopic analysis of Fdx by EPR (resonance at g = 2.03) and magnetic CD (intensity for oxidized and reduced forms and signal intensity dependence on field strength and temperature) demonstrated that Fdx binds a [3Fe-4S] iron-sulfur cluster. Potentiometric studies show that the midpoint potential for ligand-free CYP51 is -375 mV, increasing to -225 mV in the estriol-bound form. The Fdx potential is -31 mV. Fdx forms a productive electron transfer complex with CYP51 and reduces it at a rate of 3.0 min(-1) in the ligand-free form and 4.3 min(-1) in the estriol-bound form, despite a thermodynamic barrier. Steady-state analysis of a M. tuberculosis class I redox system comprising flavoprotein reductase A (FprA), Fdx, and estriol-bound CYP51 indicates heme iron reduction as a rate-limiting step.

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

“…Our data indicate an unusually high imidazole affinity (K d ϭ 1.6 M) for XplA, where this polar ligand typically has millimolar affinity for the hydrophobic active sites of most P450s (e.g. K d ϭ 11.7 mM for the M. tuberculosis CYP51B1 sterol demethylase P450) (47). Titration of the imidazole-free XplA with RDX results in a typical type I spectral shift and a much tighter K d (7.5 M) than previously reported for the 420 nm Soret form of XplA (57.9 M) (21).…”

“…Imidazole produced the expected shift to 424.5 nm (supplemental Fig. S7) with a K d of 1.6 Ϯ 0.1 M, a substantially tighter value than for other bacterial P450s, such as the Mycobacterium tuberculosis CYP121 and CYP51B1 enzymes (Ͼ50 and 11.7 mM, respectively) (47,49). The unusually high affinity for imidazole is clearly consistent with its retention as a heme ligand in XplA purification schemes involving nickel affinity chromatography.…”

Genome Mining in Sorangium cellulosum So ce56

“…Recently published examples for cytochrome P450 systems containing ferredoxins of the [3Fe-4S] cluster type are the P450mor system from Mycobacterium sp. strain HE5 (40), the CYP51 system from M. tuberculosis (41), or the CYP105D system from Streptomyces coelicolor A3(2) (45). Indeed we found the two non-[2Fe-2S] cluster ferredoxins Fdx2 and Fdx8 capable to deliver electrons from NADPH via FdR_A or FdR_B to CYP260A1 and to sustain the conversion of nootkatone to hydroxylated products.…”

“…FdR_B formed a neutral, blue semiquinone with a characteristic line width of 19 G. The line width of the FdR_A semiquinone was with 17.5 G slightly smaller but is still in the range of a neutral semiquinone (typical line width of 19 G as opposed to the anionic semiquinone with a typical line width of 14 -15 G (43)). Interestingly, not only spinach ferredoxin reductase (line width 20 G) but also the mammalian adrenodoxin reductase (line width 18 G) and its mycobacterial homologue FprA, which are both known to be components of class I cytochrome P450 systems, were reported to form neutral semiquinones (28,41,44).…”

Production of alkenes and novel secondary products by P450 OleTJE using novel H2O2‐generating fusion protein systems

“…The Bacillus megaterium P450 BM3 heme domain (BM3) and Mycobacterium tuberculosis CYP51B1 and CYP121A1 P450s were expressed and purified as described previously (20)(21)(22).…”