The cytochrome P450/P450 reductase fusion enzyme CYP505A30 from the thermophilic fungus Myceliophthora thermophila and its heme (P450) domain were expressed in Escherichia coli and purified using affinity, ion exchange, and size exclusion chromatography. CYP505A30 binds straight chain fatty acids (from ∼C10 to C20), with highest affinity for tridecanoic acid (K D = 2.7 μM). Reduced nicotinamide adenine dinucleotide phosphate is the preferred reductant for CYP505A30 (K M = 3.1 μM compared to 330 μM for reduced nicotinamide adenine dinucleotide in cytochrome c reduction). Electron paramagnetic resonance confirmed cysteine thiolate coordination of heme iron in CYP505A30 and its heme domain. Redox potentiometry revealed an unusually positive midpoint potential for reduction of the flavin adenine dinucleotide and flavin mononucleotide cofactors (E 0 ′ ∼ -118 mV), and a large increase in the CYP505A30 heme domain Fe III /Fe II redox couple (ca. 230 mV) on binding arachidonic acid substrate. This switch brings the ferric heme iron potential into the same range as that of the reductase flavins. Multiangle laser light scattering analysis revealed CYP505A30's ability to dimerize, whereas the heme domain is monomeric. These data suggest CYP505A30 may function catalytically as a dimer (as described for Bacillus megaterium P450 BM3), and that binding interactions between CYP505A30 heme domains are not required for dimer formation. CYP505A30 catalyzed hydroxylation of straight chain fatty acids at the ω-1 to ω-3 positions, with a strong preference for ω-1 over ω-3 hydroxylation in the oxidation of dodecanoic and tetradecanoic acids (88 vs 2% products and 63 vs 9% products, respectively). CYP505A30 has important structural and catalytic similarities to P450 BM3 but distinct regioselectivity of lipid substrate oxidation with potential biotechnological applications.