Kinetics and thermodynamics of CO binding to cytochrome P450nor.
The CO-binding reaction of cytochrome P450nor isolated from denitrifying fungus, Fusarium oxysporum, has been studied by using a flash photolysis method in the millisecond time domain. We obtained the CO on- and off-rate constants in the bimolecular reaction, and determined the activation free energy, enthalpy, and entropy from the temperature dependence of these rate constants. To discuss the structural characteristics of P450nor, these parameters were compared with those of other cytochrome P450s, such as cytochrome P450cam from Pseudomonas putida and myoglobin. The on-rate constant (k(on)) for P450nor is larger than those of camphor-bound P450cam [P450cam(+)], suggesting that ligand entry to the heme pocket of P450nor is sterically less restricted than that of P450cam(+). In the P450norCO complex, the IR stretching band of the iron-bound CO is observed at 1942 cm-1, which is the same position as in P450cam(+)CO. This result suggests that the heme pocket immediate to the ligand-binding site is the same size in the two enzymes, in good agreement with the observation that the equilibrium constant (K = k(on)/k(off)) is identical in P450nor and P450cam(+). On the other hand, the entropy changes in the equilibrium and the off-activation processes are smaller in P450nor than in P450cam(+). This feature could reflect the lack of a bound substrate at the active site of P450nor. These structural characteristics of P450nor are discussed in relation to its unique catalytic property, rapid NO reduction to yield N2O.
Institute of Physical and Chemical Research (RIKEN), Saitama, Japan.
Y
Y
Shiro
M
M
Kato
T
T
Iizuka
K
K
Nakahara
H
H
Shoun
D002248:Carbon Monoxide / Q000737:chemistry*; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry*; D005656:Fungal Proteins / Q000737:chemistry; D005670:Fusarium / Q000737:chemistry; D007700:Kinetics; D009569:Nitric Oxide / Q000737:chemistry; D010088:Oxidoreductases / Q000737:chemistry*; D013816:Thermodynamics
D005656:Fungal Proteins; D009569:Nitric Oxide; D002248:Carbon Monoxide; D003577:Cytochrome P-450 Enzyme System; D010088:Oxidoreductases; C080610:nitric-oxide reductase (P450)