KINETICS AND THERMODYNAMICS OF CO BINDING TO CYTOCHROME P450(NOR)

The CO-binding reaction of cytochrome P450(nor) isolated from denitrif ying fungus, Fusarium oxysporum, has been studied by using a flash pho tolysis 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 de pendence of these rate constants. To discuss the structural characteri stics of P450(nor) these parameters were compared with those of other cytochrome P450s, such as cytochrome P450(cam) from Pseudomonas putida and myoglobin. The on-rate constant (k(on)) for P450(nor) is larger t han those of camphor-bound P450(cam) [P450(cam)(+)], suggesting that l igand entry to the heme pocket of P450(nor) is sterically less restric ted than that of P450(cam)(+). In the P450(nor)CO complex, the IR stre tching band of the iron-bound CO is observed at 1942 cm(-1), which is the same position as in P450(cam)(+)CO. This result suggests that the heme pocket immediate to the ligand-binding site is the same size in t he two enzymes, in good agreement with the observation that the equili brium constant (K = k(on)/k(off)) is identical in P450(nor) and P450(c am)(+). On the other hand, the entropy changes in the equilibrium and the off-activation processes are smaller in P450(nor) than in P450(cam )(+). This feature could reflect the lack of a bound substrate at the active site of P450(nor). These structural characteristics of P450(nor ) are discussed in relation to its unique catalytic property, rapid NO reduction to yield N2O.