Spin equilibrium and O₂-binding kinetics of Mycobacterium tuberculosis CYP51 with mutations in the histidine-threonine dyad

Abstract

The acidic residues of the "acid-alcohol pair" in CYP51 enzymes are uniformly replaced with histidine. Herein, we adopt the Mycobacterium tuberculosis (mt) enzyme as a model system to investigate these residues' roles in finely tuning the heme conformation, iron spin state, and formation and decay of the oxyferrous enzyme. Properties of the mtCYP51 and the T260A, T260V, and H259A mutants were interrogated using UV-Vis and resonance Raman spectroscopies. Evidence supports that these mutations induce comprehensive changes in the heme environment. The heme iron spin states are differentially sensitive to the binding of the substrate, dihydrolanosterol (DHL). DHL and clotrimazole perturb the local environments of the heme vinyl and propionate substituents. Molecular dynamics (MD) simulations of the DHL-enzyme complexes support that the observed perturbations are attributable to changes in the DHL binding mode. Furthermore, the rates of the oxyferrous formation were measured using stopped-flow methods. These studies demonstrate that both HT mutations and DHL modulate the rates of oxyferrous formation. Paradoxically, the binding rate to the H259A mutant-DHL complex was approximately four-fold that of mtCYP51, a phenomenon that is predicted to result from the creation of an additional diffusion channel from loss of the H259-E173 ion pair in the mutant. Oxyferrous enzyme auto-oxidation rates were relatively constant, with the exception of the T260V-DHL complex. MD simulations lead us to speculate that this behavior may be attributed to the distortion of the heme macrocycle by the substrate.

Keywords: CYP51; Cytochrome P450; Molecular dynamics; Resonance Raman spectroscopy; Stopped-flow spectroscopy.

Fig. 1

Superimposition of the mtCYP51 (orange, lighter color) and CYP101 (blue, darker color) active sites illustrating the positions of the CYP51 H259–T260 dyad and CYP101 “acid–alcohol” pair (D251–T252) relative to the hemes. PDB accession numbers 2VKU and 1DZ8 for mtCYP51 and CYP101 were used, respectively.

Fig. 2

High frequency regions of the resonance Raman spectra of mtCYP51 and mutants. Spectra were collected using 406.7 nm laser excitation. Spectra of mtCYP51 (A), T260A (B), T260V (C), and H259A (D) each utilized a 10 μM protein concentration. Spectra of ligand-free proteins are depicted in blue, 50 μM DHL in green, 25 μM clotrimazole in red, 300 μM fluconazole in purple, and 300 μM ketoconazole in black.

Fig. 3

Low frequency regions of the resonance Raman spectra of mtCYP51 and mutants. Spectra were collected using 406.7 nm laser excitation. Spectra of mtCYP51 (A), T260A (B), T260V (C), and H259A (D) each utilized a 10 μM protein concentration. Spectra of ligand-free proteins are depicted in blue, 50 μM DHL in green, 25 μM clotrimazole in red, 300 μM fluconazole in purple, and 300 μM ketoconazole in black.

Fig. 4

Superimposition of the final trajectory frame of the MD simulations. (A) The binding modes of DHL in mtCYP51 (green), T260V (yellow), and H259A (purple). (B) Residues within hydrogen-bonding distance (1.5Å) of the heme propionates. MtCYP51, T260V, and H259A are in represented in green, yellow, and purple respectively.

Fig. 5

Stopped-flow reactions of CYP101 with 1 mM camphor (A) mtCYP51 (C) and mtCYP51 with 50 μM DHL (E). The corresponding spectra of oxyferrous and ferric CYP101 (B), ferric, ferrous, and, oxyferrous mtCYP51 (D) and ferric, ferrous, and, oxyferrous mtCYP51 with 50 μM DHL (F) derived from SVD analysis. An enlarged view of the Soret band maxima are illustrated in the inset. The reaction progress curves illustrating the concentrations of each species are inset into panels B, D, and F. The protein concentrations were 10 μM. Four-hundred spectral traces were collected per second.

Fig. 6

UV–Vis spectra of the ferric, ferrous, and oxyferrous states of 10 μM mtCYP51 in aqueous glycerol matrices at 80 K. Spectra were collected in 50 mM Tris–HCl (pH 8.0) with 75% (v/v) glycerol in the absence (panel A) and presence of 50 μM DHL (panel B).

Fig. 7

Implicit ligand sampling PMF maps for O₂ migration in mtCYP51, H259A, and T260V. The common O₂ migration pathways for mtCYP51, T260V and H259A are illustrated in panels A, C, and F, respectively. The prematurely terminating channel in mtCYP51 is illustrated in panel B. The second O₂ migration channel in H259A and the occupant water molecules are illustrated in panels D and E, respectively. Isosurface maps are illustrated at 2.5 kcal mol⁻¹.

Scheme 1