5WP2

1.44 Angstrom crystal structure of CYP121 from Mycobacterium tuberculosis in complex with substrate and CN

Summary for 5WP2

• Entry DOI: 10.2210/pdb5wp2/pdb
• Descriptor: Mycocyclosin synthase, PROTOPORPHYRIN IX CONTAINING FE, (3S,6S)-3,6-bis(4-hydroxybenzyl)piperazine-2,5-dione, ... (6 entities in total)
• Functional Keywords: complex, p450, oxidoreductase
• Biological source: Mycobacterium tuberculosis
• Total number of polymer chains: 1
• Total formula weight: 44527.77

Authors

Fielding, A.,Dornevil, K.,Liu, A. (deposition date: 2017-08-03, release date: 2018-05-30, Last modification date: 2023-10-04)

Primary citation

Fielding, A.J.,Dornevil, K.,Ma, L.,Davis, I.,Liu, A.
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.
J. Am. Chem. Soc., 139:17484-17499, 2017

PubMed 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 O 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 (k) but a much more stable cyanide adduct with 3 orders of magnitude slower k 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.

PubMed: 29090577
DOI: 10.1021/jacs.7b08911

Experimental method

X-RAY DIFFRACTION (1.439 Å)