2IJ4

Structure of the A264K mutant of cytochrome P450 BM3

Summary for 2IJ4

• Entry DOI: 10.2210/pdb2ij4/pdb
• Related: 1SMI 2IJ3 2IJ5 2IJ7
• Descriptor: cytochrome P450 BM3, PROTOPORPHYRIN IX CONTAINING FE (3 entities in total)
• Functional Keywords: lysine heme ligation, cytochrome p450, p450 bm3, oxidoreductase
• Biological source: Bacillus megaterium
• Cellular location: Cytoplasm (By similarity): P14779
• Total number of polymer chains: 2
• Total formula weight: 108627.37

Authors

Toogood, H.S.,Leys, D. (deposition date: 2006-09-29, release date: 2006-11-07, Last modification date: 2023-08-30)

Primary citation

Girvan, H.M.,Seward, H.E.,Toogood, H.S.,Cheesman, M.R.,Leys, D.,Munro, A.W.
Structural and spectroscopic characterization of P450 BM3 mutants with unprecedented P450 heme iron ligand sets. New heme ligation states influence conformational equilibria in P450 BM3.
J.Biol.Chem., 282:564-572, 2007

PubMed Abstract: Two novel P450 heme iron ligand sets were generated by directed mutagenesis of the flavocytochrome P450 BM3 heme domain. The A264H and A264K variants produce Cys-Fe-His and Cys-Fe-Lys axial ligand sets, which were validated structurally and characterized by spectroscopic analysis. EPR and magnetic circular dichroism (MCD) provided fingerprints defining these P450 ligand sets. Near IR MCD spectra identified ferric low spin charge-transfer bands diagnostic of the novel ligands. For the A264K mutant, this is the first report of a Cys-Fe-Lys near-IR MCD band. Crystal structure determination showed that substrate-free A264H and A264K proteins crystallize in distinct conformations, as observed previously in substrate-free and fatty acid-bound wild-type P450 forms, respectively. This, in turn, likely reflects the positioning of the I alpha helix section of the protein that is required for optimal configuration of the ligands to the heme iron. One of the monomers in the asymmetric unit of the A264H crystals was in a novel conformation with a more open substrate access route to the active site. The same species was isolated for the wildtype heme domain and represents a novel conformational state of BM3 (termed SF2). The "locking" of these distinct conformations is evident from the fact that the endogenous ligands cannot be displaced by substrate or exogenous ligands. The consequent reduction of heme domain conformational heterogeneity will be important in attempts to determine atomic structure of the full-length, multidomain flavocytochrome, and thus to understand in atomic detail interactions between its heme and reductase domains.

PubMed: 17077084
DOI: 10.1074/jbc.M607949200

Experimental method

X-RAY DIFFRACTION (2.4 Å)