Probing Ligand Exchange in the P450 Enzyme CYP121 from Mycobacterium tuberculosis: Dynamic Equilibrium of the Distal Heme Ligand as a Function of pH and Temperature

Abstract

CYP121 is a cytochrome P450 enzyme from Mycobacterium tuberculosis that catalyzes the formation of a C–C bond between the aromatic groups of its cyclodityrosine substrate (cYY). The crystal structure of CYP121 in complex with cYY reveals that the solvent-derived ligand remains bound to the ferric ion in the enzyme–substrate complex. Whereas in the generally accepted P450 mechanism, binding of the primary substrate in the active-site triggers the release of the solvent-derived ligand, priming the metal center for reduction and subsequent O2 binding. Here we employed sodium cyanide to probe the metal–ligand exchange of the enzyme and the enzyme–substrate complex. The cyano adducts were characterized by UV–vis, EPR, and ENDOR spectroscopies and X-ray crystallography. A 100-fold increase in the affinity of cyanide binding to the enzyme–substrate complex over the ligand-free enzyme was observed. The crystal structure of the [CYP121(cYY)CN] ternary complex showed a rearrangement of the substrate in the active-site, when compared to the structure of the binary [CYP121(cYY)] complex. Transient kinetic studies showed that cYY binding resulted in a lower second-order rate constant (kon (CN)) but a much more stable cyanide adduct with 3 orders of magnitude slower koff (CN) rate. A dynamic equilibrium between multiple high- and low-spin species for both the enzyme and enzyme–substrate complex was also observed, which is sensitive to changes in both pH and temperature. Our data reveal the chemical and physical properties of the solvent-derived ligand of the enzyme, which will help to understand the initial steps of the catalytic mechanism.

CW EPR parameters reveal cytochrome P450 ligand binding modes

“…Multiple complexes with the same binding mode, Figure 2S, have minor variations along the first and second principal components. The variations do not represent major differences in binding, but they could reflect different ligand-iron rotamers, variations in H-bonding networks, or different protonation states of the protein [18]. The clear distinction of binding mode by these two principal component suggests that the binding mode could be assigned solely by CW-EPR spectra.…”

“…No high spin heme was observed for any samples; this could be due in part to the fact that they were frozen in liquid nitrogen over the course of a few seconds. The freeze rate and method do seem to influence the proportion of high spin heme present, and liquid nitrogen has been reported to produce more low spin complexes [18]. All of these compounds have pharmaceutical significance.…”

“…CYP crystal structures containing drugs or substrates occasionally have binding modes that neither replace or displace the axial water. The complexes of CYP125A1 with LP10 (Figure 1) [16]; CYP121A1 with fluconazole [17]; CYP121 with cyclodityrosine [18]; and possibly CYP101A1 with 2-phenylimidazole [8] all show drug or substrate in the binding pocket with the axial water intact and forming a hydrogen-bonded bridge to the drug. The crystal structure of the complex of CYP121A1 with fluconazole is notable because it contains different mixtures of water-bridged and directly-coordinated fluconazoles in each of the six CYP121A molecules that make up the asymmetric unit, suggesting that the binding energies are so similar that the two binding modes coexist in the crystal [17].…”

Formation and Cleavage of C–C Bonds by Enzymatic Oxidation–Reduction Reactions

“…C-C diradical coupling in the synthesis of dicyclotyrosine by P450 121, supported by peracetic acid in a model system. 79,80 PCET: proton-coupled electron transfer.…”

Anti-Tubercular Properties of 4-Amino-5-(4-Fluoro-3- Phenoxyphenyl)-4H-1,2,4-Triazole-3-Thiol and Its Schiff Bases: Computational Input and Molecular Dynamics

“…Triazole thiol derivatives are associated with strong antibacterial and anti-TB actions [41,49,65,74]. In order to identify the potential target of our newly synthesized parent compound 1 and its Schiff bases 2, 3, and 4, a dataset was constructed comprising the most common molecular targets for triazole derivatives from the previously published literature [58][59][60][61][62][63]. The 3D-Crystal structures of target proteins and their co-crystal structures (Figure 3) were procured from the Protein Data Bank (PDB) as shown in Table 3.…”