Structural and spectroscopic analysis of the F393H mutant of flavocytochrome p450 BM3

In the preceding paper in this issue [Ost, T. W. B., Miles, C. S., Munro, A . W., Murdoch, J., Reid, G. A., and Chapman, S. K. (2001) Biochemistry 40, 13421-13429], we have established that the primary role of the phylogenetic ally conserved phenylalanine in flavocytochrome P450 BM3 (F393) is to contr ol the thermodynamic properties of the heme iron, so as to optimize electro n-transfer both to the iron (from the flavin redox partner) and onto molecu lar oxygen. In this paper, we report a detailed study of the F393H mutant e nzyme, designed to probe the structural, spectroscopic, and metabolic profi le of the enzyme in an attempt to identify the factors responsible for caus ing the changes. The heme domain structure of the F393H mutant has been sol ved to 2.0 Angstrom resolution and demonstrates that the histidine replaces the phenylalanine in almost exactly the same conformation. A solvent water molecule is hydrogen bonded to the histidine, but there appears to be litt le other gross alteration in the environment of the heme. The F393H mutant displays an identical ferric EPR spectrum to wild-type, implying that the d egree of splitting of the iron d orbitals is unaffected by the substitution , however, the overall energy of the d-orbitals have changed relative to ea ch other. Magnetic CD studies show that the near-IR transition, diagnostic of heme ligation state, is red-shifted by 40 nn in F393H relative to wild-t ype P450 BM3, probably reflecting alteration in the strength of the iron-cy steinate bond. Studies of the catalytic turnover of fatty acid (myristate) confirms NADPH oxidation is tightly coupled to fatty acid oxidation in F393 H, with a product profile very similar to wild-type. The results indicate t hat gross conformational changes do not account for the perturbations in th e electronic features of the P450 BM3 heme system and that the structural e nvironment on the proximal side of the P450 heme must be conformationally c onserved in order to optimize catalytic function.