Phenylalanine 393 exerts thermodynamic control over the heme of flavocytochrome P450BM3

被引:101
作者
Ost, TWB
Miles, CS
Munro, AW
Murdoch, J
Reid, GA
Chapman, SK
机构
[1] Univ Edinburgh, Dept Chem, Edinburgh EH9 3JJ, Midlothian, Scotland
[2] Univ Edinburgh, Inst Cell & Mol Biol, Edinburgh EH9 3JT, Midlothian, Scotland
[3] Univ Leicester, Dept Biochem, Leicester LE1 7RH, Leics, England
关键词
D O I
10.1021/bi010716m
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Site-directed mutants of the phylogenetically conserved phenylalanine residue F393 were constructed in flavocytochrome P450 BM3 from Bacillus megaterium. The high degree of conservation of this residue in the P450 superfamily and its proximity to the heme (and its ligand Cys400) infers an essential role in P450 activity. Extensive kinetic and thermodynamic characterization of mutant enzymes F393A, F393H, and F393Y highlighted significant differences from wild-type P450 BM3. All enzymes expressed to high levels and contained their full complement of heme. While the reduction and subsequent treatment of the mutant P450s with carbon monoxide led to the formation of the characteristic P450 spectra in all cases, the absolute position of the Soret absorption varied across the series WT/F393Y (449 nm), F393H (445 nm), and F393A (444 nm). Steady-state turnover rates with both laurate and arachidonate showed the trend WT > F393Y much greater than F393H > F393A. Conversely, the trend in the pre-steady-state flavin-to-heme electron transfer was the reverse of the steady-state scenario, with rates varying F393A > F393H much greater than F393Y approximate to wild-type. These data are consistent with the more positive substrate-free [-312 mV (F393A), -332 mV (F393H)] and substrate-bound [-151 mV (F393A), -176 mV (F393H)] reduction potentials of F393A and F393H heme domains, favoring the stabilization of the ferrous-form in the mutant P450s relative to wild-type. Elevation of the heme iron reduction potential in the F393A and F393H mutants facilitates faster electron transfer to the heme. This results in a decrease in the driving force for oxygen reduction by the ferrous heme iron, so explaining lower overall turnover of the mutant P450s. We postulate that the nature of the residue at position 393 is important in controlling the delicate equilibrium observed in P450s, whereby a tradeoff is established between the rate of heme reduction and the rate at which the ferrous heme can bind and, subsequently, reduce molecular oxygen.
引用
收藏
页码:13421 / 13429
页数:9
相关论文
共 29 条
[1]   Electron transfer, oxygen binding, and nitric oxide feedback inhibition in endothelial nitric-oxide synthase [J].
Abu-Soud, HM ;
Ichimori, K ;
Presta, A ;
Stuehr, DJ .
JOURNAL OF BIOLOGICAL CHEMISTRY, 2000, 275 (23) :17349-17357
[2]   Tryptophan 409 controls the activity of neuronal nitric-oxide synthase by regulating nitric oxide feedback inhibition [J].
Adak, S ;
Crooks, C ;
Wang, Q ;
Crane, BR ;
Tainer, JA ;
Getzoff, ED ;
Stuehr, DJ .
JOURNAL OF BIOLOGICAL CHEMISTRY, 1999, 274 (38) :26907-26911
[3]  
ANDERSSON S, 1989, J BIOL CHEM, V264, P8222
[4]   Spectral properties of the oxyferrous complex of the heme domain of cytochrome P450BM-3 (CYP102) [J].
Bec, N ;
Anzenbacher, P ;
Anzenbacherová, E ;
Gorren, ACF ;
Munro, AW ;
Lange, R .
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 1999, 266 (01) :187-189
[5]   HUMAN CHOLESTEROL SIDE-CHAIN CLEAVAGE ENZYME, P450SCC - CDNA CLONING, ASSIGNMENT OF THE GENE TO CHROMOSOME-15, AND EXPRESSION IN THE PLACENTA [J].
CHUNG, B ;
MATTESON, KJ ;
VOUTILAINEN, R ;
MOHANDAS, TK ;
MILLER, WL .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1986, 83 (23) :8962-8966
[6]   Redox control of the catalytic cycle of flavocytochrome P-450 BM3 [J].
Daff, SN ;
Chapman, SK ;
Turner, KL ;
Holt, RA ;
Govindaraj, S ;
Poulos, TL ;
Munro, AW .
BIOCHEMISTRY, 1997, 36 (45) :13816-13823
[7]  
Dutton P L, 1978, Methods Enzymol, V54, P411
[8]   ISOLATION AND SEQUENCE OF CDNA-ENCODING A CYTOCHROME-P-450 FROM AN INSECTICIDE-RESISTANT STRAIN OF THE HOUSE-FLY, MUSCA-DOMESTICA [J].
FEYEREISEN, R ;
KOENER, JF ;
FARNSWORTH, DE ;
NEBERT, DW .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1989, 86 (05) :1465-1469
[10]   Crystal structures of zinc-free and -bound heme domain of human inducible nitric-oxide synthase - Implications for dimer stability and comparison with endothelial nitric-oxide synthase [J].
Li, HY ;
Raman, CS ;
Glaser, CB ;
Blasko, E ;
Young, TA ;
Parkinson, JF ;
Whitlow, M ;
Poulos, TL .
JOURNAL OF BIOLOGICAL CHEMISTRY, 1999, 274 (30) :21276-21284