title: 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.
authors.affiliation: Department of Physiology and Biophysics and The Massey Cancer Center, Virginia Commonwealth University School of Medicine, Richmond, VA 23219, United States.
authors.forename: Gareth K
authors.initials: GK
authors.lastname: Jennings
authors.affiliation: Department of Physiology and Biophysics and The Massey Cancer Center, Virginia Commonwealth University School of Medicine, Richmond, VA 23219, United States.
authors.forename: Anuja
authors.initials: A
authors.lastname: Modi
authors.affiliation: Department of Physiology and Biophysics and The Massey Cancer Center, Virginia Commonwealth University School of Medicine, Richmond, VA 23219, United States.
authors.forename: Justin E
authors.initials: JE
authors.lastname: Elenewski
authors.affiliation: Department of Physiology and Biophysics and The Massey Cancer Center, Virginia Commonwealth University School of Medicine, Richmond, VA 23219, United States.
authors.forename: Caroline M
authors.initials: CM
authors.lastname: Ritchie
authors.affiliation: Department of Physiology and Biophysics and The Massey Cancer Center, Virginia Commonwealth University School of Medicine, Richmond, VA 23219, United States.
authors.forename: Thuy
authors.initials: T
authors.lastname: Nguyen
authors.affiliation: Department of Physiology and Biophysics and The Massey Cancer Center, Virginia Commonwealth University School of Medicine, Richmond, VA 23219, United States.
authors.forename: Keith C
authors.initials: KC
authors.lastname: Ellis
authors.affiliation: Department of Physiology and Biophysics and The Massey Cancer Center, Virginia Commonwealth University School of Medicine, Richmond, VA 23219, United States. Electronic address: jchackett@vcu.edu.
authors.forename: John C
authors.initials: JC
authors.lastname: Hackett
mesh_terms: D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D020134:Catalytic Domain; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000235:genetics; D006639:Histidine / Q000737:chemistry / Q000235:genetics; D007700:Kinetics; D007810:Lanosterol / Q000031:analogs & derivatives / Q000737:chemistry; D056004:Molecular Dynamics Simulation; D016297:Mutagenesis, Site-Directed; D009169:Mycobacterium tuberculosis / Q000201:enzymology*; D010100:Oxygen / Q000737:chemistry*; D011485:Protein Binding; D013059:Spectrum Analysis, Raman; D013912:Threonine / Q000737:chemistry / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D007810:Lanosterol; D013912:Threonine; D006639:Histidine; C015316:lanostenol; D003577:Cytochrome P-450 Enzyme System; C501935:cytochrome P-450 CYP51, Mycobacterium tuberculosis; D010100:Oxygen
keywords: CYP51; Cytochrome P450; Molecular dynamics; Resonance Raman spectroscopy; Stopped-flow spectroscopy