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 phylogenetically conserved phenylalanine in flavocytochrome P450 BM3 (F393) is to
control the thermodynamic properties of the heme iron, so as to optimize electron-transfer both to the
iron (from the flavin redox partner) and onto molecular oxygen. In this paper, we report a detailed study
of the F393H mutant enzyme, designed to probe the structural, spectroscopic, and metabolic profile of
the enzyme in an attempt to identify the factors responsible for causing the changes. The heme domain
structure of the F393H mutant has been solved to 2.0 Å 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 little other gross alteration in the environment of the
heme. The F393H mutant displays an identical ferric EPR spectrum to wild-type, implying that the degree
of splitting of the iron d orbitals is unaffected by the substitution, however, the overall energy of the
d-orbitals have changed relative to each other. Magnetic CD studies show that the near-IR transition,
diagnostic of heme ligation state, is red-shifted by 40 nm in F393H relative to wild-type P450 BM3,
probably reflecting alteration in the strength of the iron-cysteinate bond. Studies of the catalytic turnover
of fatty acid (myristate) confirms NADPH oxidation is tightly coupled to fatty acid oxidation in F393H,
with a product profile very similar to wild-type. The results indicate that gross conformational changes
do not account for the perturbations in the electronic features of the P450 BM3 heme system and that the
structural environment on the proximal side of the P450 heme must be conformationally conserved in
order to optimize catalytic function.
