The catalytic mechanism of cytochrome P450 BM3 involves a 6 Å movement of the bound substrate on reduction

The nitrogenous -acceptor ligand 4-cyanopyridine (4CNPy) exhibits reversible ligation to ferrous heme in the flavocytochrome P450 BM3 (K d ‫؍‬ 1.8 M for wild type P450 BM3) via its pyridine ring nitrogen. The reduced P450 -4CNPy adduct displays unusual spectral properties that provide a useful spectroscopic handle to probe particular aspects of this P450. 4CNPy is competitively displaced upon substrate binding, allowing a convenient route to the determination of substrate dissociation constants for ferrous P450 highlighting an increase in P450 substrate affinity on heme reduction. For wild type P450 BM3, K d (red)(laurate) ‫؍‬ 82.4 M (cf. K d (ox) ‫؍‬ 364 M). In addition, an unusual spectral feature in the red region of the absorption spectrum of the reduced P450 -4CNPy adduct is observed that can be assigned as a metal-to-ligand charge transfer (MLCT). It was discovered that the energy of this MLCT varies linearly with respect to the P450 heme reduction potential. By studying the energy of this MLCT for a series of BM3 active site mutants with differing reduction potential (E m ), the relationship E MLCT ‫؍‬ (3.53 ؋ E m ) ؉ 17,005 cm ؊1 was derived. The use of this ligand thus provides a quick and accurate method for predicting the heme reduction potentials of a series of P450 BM3 mutations using visible spectroscopy, without the requirement for redox potentiometry.

Section: Discussionmentioning

confidence: 54%

mentioning

confidence: 99%

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4-Cyanopyridine, a Versatile Spectroscopic Probe for Cytochrome P450 BM3

Ost

Clark

Anderson

et al. 2004

“…NMR studies of the substrate-bound form of ferrous, fatty acid-bound wild-type P450 BM3 are consistent with a significant reorientation of the substrate in this enzyme form (27). It is clear, however, that, upon binding of the fatty acid analog N-palmitoylglycine, the surroundings of site L become more hydrophobic, decreasing the water affinity and ultimately shifting the water molecule to predominantly occupy site H. Spectroscopic studies of wild-type P450 BM3 at catalytically relevant temperatures (by both electronic absorption and resonance Raman) have shown that, even in the presence of apparently saturating concentrations of substrate, an equilibrium exists between the high-spin five-coordinate and the low-spin six-coordinate states of the heme iron.…”

Section: Resultsmentioning

confidence: 73%

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

Joyce¹,

Girvan²,

Munro³

et al. 2004

The multidomain fatty-acid hydroxylase flavocytochrome P450 BM3 has been studied as a paradigm model for eukaryotic microsomal P450 enzymes because of its homology to eukaryotic family 4 P450 enzymes and its use of a eukaryotic-like diflavin reductase redox partner. High-resolution crystal structures have led to the proposal that substrate-induced conformational changes lead to removal of water as the sixth ligand to the heme iron. Concomitant changes in the heme iron spin state and heme iron reduction potential help to trigger electron transfer from the reductase and to initiate catalysis. Surprisingly, the crystal structure of the substrate-free A264E heme domain mutant reveals the enzyme to be in the conformation observed for substrate-bound wild-type P450, but with the iron in the low-spin state. This provides strong evidence that the spin-state shift observed upon substrate binding in wildtype P450 BM3 not only is caused indirectly by structural changes in the protein, but is a direct consequence of the presence of the substrate itself, similar to what has been observed for P450cam. The crystal structure of the palmitoleate-bound A264E mutant reveals that substrate binding promotes heme ligation by Glu 264 , with little other difference from the palmitoleate-bound wildtype structure observable. Despite having a protein-derived sixth heme ligand in the substrate-bound form, the A264E mutant is catalytically active, providing further indication for structural rearrangement of the active site upon reduction of the heme iron, including displacement of the glutamate ligand to allow binding of dioxygen.

“…In reporting their structure of the palmitoleatebound form of the wild-type P450 BM3 heme domain, Li and Poulos (65) noted that the distance between the -carbon of the bound substrate and the heme iron is too great for oxidative attack of the substrate and that further structural change resulting in repositioning of the substrate closer to the heme is required following reduction of the ferric iron. NMR studies of the ferric and ferrous P450 enzymes indicate that there should be a 6-Å movement of substrate subsequent to heme iron reduction (70). Thus, it appears clear that the Cys-Glu coordination is broken following heme iron reduction, enabling binding of oxygen and catalysis to ensue, albeit with lower catalytic rate than in the wild-type enzyme.…”

Section: Discussionmentioning

confidence: 99%

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

Girvan

Marshall

Lawson

et al. 2004

101

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.,