4-cyanopyridine, a versatile spectroscopic probe for cytochrome P450 BM3.
The nitrogenous pi -acceptor ligand 4-cyanopyridine (4CNPy) exhibits reversible ligation to ferrous heme in the flavocytochrome P450 BM3 (Kd=1.8 microm 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, Kd(red)(laurate)=82.4 microm (cf. Kd(ox)=364 microm). 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 (Em), the relationship EMLCT + (3.53 x = Em 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.
Ost
Tobias W B
TW
Department of Chemistry, University of Edinburgh, West Mains Road, Edinburgh EH9 3JJ, Scotland, United Kingdom.
Clark
Jonathan P
JP
Anderson
J L Ross
JL
Yellowlees
Lesley J
LJ
Daff
Simon
S
Chapman
Stephen K
SK
D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D001665:Binding Sites; D001667:Binding, Competitive; D004396:Coloring Agents / Q000494:pharmacology*; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000235:genetics; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000378:metabolism; D005227:Fatty Acids / Q000737:chemistry; D006418:Heme / Q000737:chemistry; D007501:Iron / Q000737:chemistry; D007700:Kinetics; D008024:Ligands; D006899:Mixed Function Oxygenases / Q000737:chemistry* / Q000235:genetics; D008956:Models, Chemical; D009154:Mutation; D009251:NADPH-Ferrihemoprotein Reductase; D009570:Nitriles / Q000737:chemistry / Q000494:pharmacology*; D010084:Oxidation-Reduction; D017354:Point Mutation; D011485:Protein Binding; D011725:Pyridines / Q000737:chemistry / Q000494:pharmacology*; D013053:Spectrophotometry / Q000379:methods*; D013379:Substrate Specificity; D013816:Thermodynamics; D014466:Ultraviolet Rays
D001426:Bacterial Proteins; D004396:Coloring Agents; D005227:Fatty Acids; D008024:Ligands; D009570:Nitriles; D011725:Pyridines; D006418:Heme; D003577:Cytochrome P-450 Enzyme System; C494727:4-cyanopyridine; D007501:Iron; D006899:Mixed Function Oxygenases; D009251:NADPH-Ferrihemoprotein Reductase; C052423:flavocytochrome P450 BM3 monoxygenases