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In flavocytochrome P450 BM3 there are several active site residues that are highly conserved throughout the P450 superfamily. Of these, a phenylalanine (Phe393) has been shown to modulate heme reduction potential through interactions with the implicitly conserved heme-ligand cysteine. In addition, a distal threonine (Thr268) has been implicated in a variety of roles including proton donation, oxygen activation and substrate recognition. Substrate binding in P450 BM3 causes a shift in the spin state from low- to high-spin. This change in spin-state is accompanied by a positive shift in the reduction potential (DeltaE(m) [WT+arachidonate (120 microM)]=+138 mV). Substitution of Thr268 by an alanine or asparagine residue causes a significant decrease in the ability of the enzyme to generate the high-spin complex via substrate binding and consequently leads to a decrease in the substrate-induced potential shift (DeltaE(m) [T268A+arachidonate (120 microM)]=+73 mV, DeltaE(m) [T268N+arachidonate (120 microM)]=+9 mV). Rate constants for the first electron transfer and for oxy-ferrous decay were measured by pre-steady-state stopped-flow kinetics and found to be almost entirely dependant on the heme reduction potential. More positive reduction potentials lead to enhanced rate constants for heme reduction and more stable oxy-ferrous species. In addition, substitutions of the threonine lead to an increase in the production of hydrogen peroxide in preference to hydroxylated product. These results suggest an important role for this active site threonine in substrate recognition and in maintaining an efficiently functioning enzyme. However, the dependence of the rate constants for oxy-ferrous decay on reduction potential raises some questions as to the importance of Thr268 in iron-oxo stabilisation.

Nitric oxide synthase (NOS) has an oxygenase domain with a thiol-coordinated heme active side similar to cytochrome P450. In contrast to cytochrome P450, however, conserved aromatic amino acids are situated in the heme proximal side of NOS. For example, in endothelial NOS (eNOS), the indole-ring nitrogen of Trp180 hydrogen-binds to the thiol of Cys186, the internal axial ligand to the heme. And, the aromatic side chain of Trp192 forms a bridge between this residue and the protein. Trp180 and Trp192 of eNOS correspond to Trp409 and Trp421 of neuronal NOS (nNOS), respectively. In order to understand the roles of the aromatic amino acids in catalysis, we generated Trp409His, Trp409Leu, Trp421His and Trp421Leu mutants of nNOS and determined their catalytic parameters. The Trp409Leu mutant was very poorly expressed in E. coli and was easily denatured during purification procedures. The NO formation activities of the Trp409His and Trp421Leu mutants were 11 and 25 micromol/min per micromol heme, respectively, and are lower than that (44 micromol/min per micromol heme) of the wild type. The activity (46 micromol/min per micromol heme) of the Trp421His mutant was comparable to that of the wild-type enzyme. However, NADPH oxidation rates of Trp421His (230 micromol/min per micromol heme) and Trp421Leu (104 micromol/min per microol heme) in the presence of L-Arg were much larger than those observed for the wild type (65 micromol/min per micromol heme) and the Trp409His mutant (43 micromol/min per micromol heme). The cytochrome c reduction rate of the Trp421His mutant was 6-fold larger than that of the wild type. The heme reduction rate with NADPH for the Trp421His mutant (0.09 min(-1)) was much lower than that (1.0 min(-1)) of the wild type. Taken together, it appears that Trp421 may be involved in inter-domain/inter-subunit electron transfer reactions.