| Summary: | The self sufficient class III monooxygenase, flavocytochrome P450 BM3, from <i>Bacillus megaterium</i>, is a heme containing redox enzyme that catalyses the hydroxylation of long-chain fatty acids. Flavocytochrome P450 BM3 contains several active-site residues that are highly conserved throughout the P450 superfamily. Of these, a phenylalanine (Phe393) has been shown to modulate the heme reduction potential (E<sub>m</sub>) across a 200 mV range via 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 (ΔE<sub>m</sub>[WT + arachidonate (120 μM)] = +138 mV). Substitution of Thr268 by alanine or asparagines 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 (ΔE<sub>m</sub>[T268A + arachidonate (120 μM)] = +73 mV, ΔE<sub>m</sub> {T268N + arachidonate (120 μM)] = +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. These results suggest an important role for this active-site threonine in substrate recognition and in maintaining an efficiently functioning enzyme.
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