PMID- 15364917
OWN - NLM
STAT- MEDLINE
DCOM- 20050111
LR  - 20210206
IS  - 0021-9258 (Print)
IS  - 0021-9258 (Linking)
VI  - 279
IP  - 47
DP  - 2004 Nov 19
TI  - 4-cyanopyridine, a versatile spectroscopic probe for cytochrome P450 BM3.
PG  - 48876-82
AB  - 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.
FAU - Ost, Tobias W B
AU  - Ost TW
AD  - Department of Chemistry, University of Edinburgh, West Mains Road, Edinburgh EH9 
      3JJ, Scotland, United Kingdom.
FAU - Clark, Jonathan P
AU  - Clark JP
FAU - Anderson, J L Ross
AU  - Anderson JL
FAU - Yellowlees, Lesley J
AU  - Yellowlees LJ
FAU - Daff, Simon
AU  - Daff S
FAU - Chapman, Stephen K
AU  - Chapman SK
LA  - eng
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
DEP - 20040910
PL  - United States
TA  - J Biol Chem
JT  - The Journal of biological chemistry
JID - 2985121R
RN  - 0 (Bacterial Proteins)
RN  - 0 (Coloring Agents)
RN  - 0 (Fatty Acids)
RN  - 0 (Ligands)
RN  - 0 (Nitriles)
RN  - 0 (Pyridines)
RN  - 42VZT0U6YR (Heme)
RN  - 9035-51-2 (Cytochrome P-450 Enzyme System)
RN  - BY3226D010 (4-cyanopyridine)
RN  - E1UOL152H7 (Iron)
RN  - EC 1.- (Mixed Function Oxygenases)
RN  - EC 1.6.2.4 (NADPH-Ferrihemoprotein Reductase)
RN  - EC 1.6.2.4 (flavocytochrome P450 BM3 monoxygenases)
SB  - IM
MH  - Bacterial Proteins/*chemistry/genetics
MH  - Binding Sites
MH  - Binding, Competitive
MH  - Coloring Agents/*pharmacology
MH  - Cytochrome P-450 Enzyme System/*chemistry/genetics
MH  - Dose-Response Relationship, Drug
MH  - Escherichia coli/metabolism
MH  - Fatty Acids/chemistry
MH  - Heme/chemistry
MH  - Iron/chemistry
MH  - Kinetics
MH  - Ligands
MH  - Mixed Function Oxygenases/*chemistry/genetics
MH  - Models, Chemical
MH  - Mutation
MH  - NADPH-Ferrihemoprotein Reductase
MH  - Nitriles/chemistry/*pharmacology
MH  - Oxidation-Reduction
MH  - Point Mutation
MH  - Protein Binding
MH  - Pyridines/chemistry/*pharmacology
MH  - Spectrophotometry/*methods
MH  - Substrate Specificity
MH  - Thermodynamics
MH  - Ultraviolet Rays
EDAT- 2004/09/15 05:00
MHDA- 2005/01/12 09:00
CRDT- 2004/09/15 05:00
PHST- 2004/09/15 05:00 [pubmed]
PHST- 2005/01/12 09:00 [medline]
PHST- 2004/09/15 05:00 [entrez]
AID - S0021-9258(19)32275-6 [pii]
AID - 10.1074/jbc.M408601200 [doi]
PST - ppublish
SO  - J Biol Chem. 2004 Nov 19;279(47):48876-82. doi: 10.1074/jbc.M408601200. Epub 2004 
      Sep 10.