Thermodynamic and Kinetic Studies on the Binding of Nitric Oxide to a New Enzyme Mimic of Cytochrome P450

Abstract

A new model for the P450 enzyme carrying a SO(3)(-) ligand coordinated to iron(III) (complex 2) reversibly binds NO to yield the nitrosyl adduct. The rate constant for NO binding to 2 in toluene is of the same order of magnitude as that found for the nitrosylation of the native, substrate-bound form of P450(cam) (E.S-P450(cam)). Large and negative activation entropy and activation volume values for the binding of NO to complex 2 support a mechanism that is dominated by bond formation with concomitant iron spin change from S = (5)/(2) to S = 0, as proposed for the reaction between NO and E.S-P450(cam). In contrast, the dissociation of NO from 2(NO) was found to be several orders of magnitude faster than the corresponding reaction for the E.S-P450(cam)/NO system. In a coordinating solvent such as methanol, the alcohol coordinates to iron(III) of 2 at the distal position, generating a six-coordinate, high-spin species 5. The reaction of NO with 5 in methanol was found to be much slower in comparison to the nitrosylation reaction of 2 in toluene. This behavior can be explained in terms of a mechanism in which methanol must be displaced during Fe-NO bond formation. The thermodynamic and kinetic data for NO binding to the new model complexes of P450 (2 and 5) are discussed in reference to earlier results obtained for closely related nitrosylation reactions of cytochrome P450(cam) (in the presence and in the absence of the substrate) and a thiolate-ligated iron(III) model complex.

Mechanistic Studies on the Activation of NO by Iron and Cobalt Complexes

“…Since the Fe III -heme centre in substrate-bound P450 cam is five-coordinate, formation of an Fe-NO bond does not require initial displacement of a water molecule, and therefore the ligation cannot be limited by the rate of H 2 O dissociation as was observed for the reaction with substrate-free P450 cam . These features are consistent with the associatively activated mechanism in which an encounter complex, {(P)Fe III ʈNO}, is formed prior to NO bond formation, as was also proposed for the reaction of NO with [44] (E·S-P450 cam ) [44] in methanol [62] in toluene [67] in methanol [67] ferroheme proteins [56] and their model complexes [32b,33c] or with the water-soluble monohydroxo iron(III) porphyrin models. [40,41,43] The value of the second-order rate constant for the binding of NO to camphor-bound P450 cam is relatively small relative to the rate constants known for the diffusion-controlled reactions in water.…”

“…Volume profiles for reversible NO binding to (a) the substrate-free form of P450 cam [44] and (b) complex 4 in methanol. [67] tein and the heme pocket to be more rigid and less compressible [57] ) and/or the much larger reorganization of spin multiplicity upon NO binding to substrate-bound P450 cam (from S = 5/2 to S = 0) than that observed for the substratefree analogue (from S = 1/2 to S = 0).…”

“…[67] In toluene, the five-coordinate, high-spin complex 3 reminiscent of the camphor-bound P450 cam is formed, whereas in methanol the six-coordinate complex 4 (see Figure 11) is produced, which resembles the resting state of P450 cam . As can be seen from Table 4, model complex 3, although having no protein pocket and possessing a SO 3 -group as proximal ligand, displays a NO binding rate constant in a non-coordinating solvent that is similar to that measured for the NO association reaction of camphor-bound P450 cam .…”

“…[67] In methanol, complex 4 is a six-coordinate, high-spin species. The presence of methanol coordinated to iron(III) slows down the binding of NO to 4 by about a factor of 10 relative to the binding of NO to 3, which lacks the sixth ligand (Table 4).…”

Application of High Pressure in the Elucidation of Inorganic and Bioinorganic Reaction Mechanisms

“…The report displayed the volume profile of the reversible binding of NO to complex 5 in methanol in tandem with that for NO binding to the substrate-free form of P450 cam and presented a detailed discussion of the contrasting and in other aspects similar properties. 332 The latter profile is in Figure 7.16a, with the former presented in Figure 7.17. Recently, a related enzyme mimic of cytochrome P450 has been used as a catalyst in the epoxidation reaction of cis-stilbene with m-chloroperoxybenzoic acid.…”