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Publication Abstract from PubMed

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.

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.,Fielding AJ, Dornevil K, Ma L, Davis I, Liu A J Am Chem Soc. 2017 Dec 6;139(48):17484-17499. doi: 10.1021/jacs.7b08911. Epub, 2017 Nov 20. PMID:29090577^[1]

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.