Solution phase refinement of active site structure using 2D NMR and judiciously 13C-labeled cytochrome P450

The Cytochrome P450 (CYP450) superfamily has been the subject of intense research for over six decades. Here the HU227 strain of E. coli, lacking the δ-aminolevulinic acid (δ-ALA) synthase gene, was employed, along with [5-13C] δ-ALA, in the heterologous expression of P450cam harboring a prosthetic group labeled with 13C at the four methine carbons (Cm) and pyrrole Cα positions. The product was utilized as a proof of principle strategy for defining and refining solution phase active site structure in cytochrome P450cam, providing proton-to-proton distances from 13CmH to protons on bound substrate or nearby amino acid residues, using short mixing time 2D or 3D NOESY-HMQC methods. The results reveal the interesting finding that 2D 13C-filtered NOESY-HMQC can be used to obtain distances between protons on labeled 13C to positions of protons nearby in the active site, confirming the utility of this NMR-based approach to probing active site structure under physiological conditions. Such 13C-heme-filtered NOE data complement X-ray crystallographic and T1-based NMR measurements; and, may also be of potentially significant utility in furnishing experimental distance constraints in validations of docking routines commonly employed for determining the relative affinities and binding orientations of drug candidates with CYP450s. Crosspeaks showing protons nearby 13C -labeled methine protons. [Display omitted] •Synthesis of aminolevulinic acid, a heme precursor.•Use of E. coli strain, HU227 and adding aminolevulinic acid to make labeled heme.•Use of NOE to get distances between protons on labeled positions and on substrate.•NOE crosspeaks observed for protons close to each other, ≤ 5 Å through space.•Good agreement between NOE distances and X-ray crystallographic data.•Such experimentally-derived parameters important for refining docking procedures.