Flavocytochrome P450 BM3 Mutant A264E Undergoes Substrate-dependent Formation of a Novel Heme Iron Ligand Set

Abstract

A conserved glutamate covalently attaches the heme to the protein backbone of eukaryotic CYP4 P450 enzymes. In the related Bacillus megaterium P450 BM3, the corresponding residue is Ala 264 . The A264E mutant was generated and characterized by kinetic and spectroscopic methods. A264E has an altered absorption spectrum compared with the wild-type enzyme (Soret maximum at ϳ420.5 nm). Fatty acid substrates produced an inhibitor-like spectral change, with the Soret band shifting to 426 nm. Optical titrations with long-chain fatty acids indicated higher affinity for A264E over the wild-type enzyme. The heme iron midpoint reduction potential in substrate-free A264E is more positive than that in wild-type P450 BM3 and was not changed upon substrate binding. EPR, resonance Raman, and magnetic CD spectroscopies indicated that A264E remains in the low-spin state upon substrate binding, unlike wild-type P450 BM3. EPR spectroscopy showed two major species in substrate-free A264E. The first has normal Cys-aqua iron ligation. The second resembles formateligated P450cam. Saturation with fatty acid increased the population of the latter species, suggesting that substrate forces on the glutamate to promote a Cys-Glu ligand set, present in lower amounts in the substratefree enzyme. A novel charge-transfer transition in the near-infrared magnetic CD spectrum provides a spectroscopic signature characteristic of the new A264E heme iron ligation state. A264E retains oxygenase activity, despite glutamate coordination of the iron, indicating that structural rearrangements occur following heme iron reduction to allow dioxygen binding. Glutamate coordination of the heme iron is confirmed by structural studies of the A264E mutant (Joyce, M. G.,

Unusual Spectroscopic and Ligand Binding Properties of the Cytochrome P450-Flavodoxin Fusion Enzyme XplA

“…S9), a slightly longer wavelength than has been observed for other P450s analyzed to date (e.g. ϳ1080 nm for P450 BM3) (32). This suggests that the t 2 g heme iron D-orbitals are closer in energy to the porphyrin orbitals in XplA than is typical in the Cys thiolate-coordinated P450s, possibly due to the electrostatic environment of the thiolate ligand.…”

“…The polar imidazole is usually a weak inhibitory ligand for P450s, although substituted imidazoles can be much tighter binding as a consequence of their more apolar nature and favorable interactions of their substituent groups with active site amino acid side chains. For instance, the P450 BM3 K d for imidazole is several mM, whereas the K d values for 4-phenylimidazole and the fatty acid derivative 12-(imidazolyl)dodecanoic acid are 0.85 and 8 M, respectively (32,43). In order to explore further whether imidazole might be retained by XplA upon protein purification, we performed more extensive dialysis with several buffer changes following the Ni-NTA chromatography step.…”

Functional expression and characterization of cytochrome P450 52A21 from Candida albicans

“…However, it is significant that the conserved Glu residue responsible for covalent attachment of the heme to the protein in the P450 4A family is not found in CYP52A21 (Fig. 1) [27]. Indeed, heme staining by adding 3,3',5,5'-tetramethylbenzidine and H 2 O 2 to an SDS-Page gel showed that CYP52A21 does not have a covalently bound heme, in contrast to CYP4A1 which, as expected, does contain the proteinbound heme (supplementary Fig.…”

A Single Mutation in Cytochrome P450 BM3 Induces the Conformational Rearrangement Seen upon Substrate Binding in the Wild-type Enzyme

“…A final purification step by fast protein liquid chromatography using Q-Sepharose resin (under the same conditions as used for the lowpressure chromatographic purification described in the accompanying article (17)) was used to produce homogeneous enzyme for crystallographic studies.…”

“…To investigate the possibility of creating a similar protein-heme link in the related P450 BM3 heme domain, the A264E mutant was created. Although no covalent modification of the heme was observed, this mutant has several unique features (see accompanying article (17)). In the fatty acid-free form, Glu 264 ligates the heme iron in a proportion of the molecules, creating a novel thiolate-carboxylate ligation that is pushed toward full ligation by binding of the substrate.…”