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CYP119 from Sulfolobus solfataricus is the first well-characterized thermophilic cytochrome P450 enzyme. The endogenous substrate for this enzyme is not known but it hydroxylates lauric acid in a reaction supported by surrogate mesophilic electron donors. However, reconstitution of a high-temperature catalytic system requires identification of the normal thermophilic electron donor partners of CYP119. Here, we describe cloning, expression in Escherichia coli, and characterization of the requisite electron donor partners from S. solfataricus. One is a thermostable ferredoxin and the second a 2-oxoacid-ferredoxin oxidoreductase that utilizes pyruvic acid rather than NAD(P)H as the source of reducing equivalents. CYP119 is the only cytochrome P450 to date known to obtain electrons from a non-NAD(P)H-dependent protein. The two thermophilic partners have been used to reconstitute a catalytic system that hydroxylates lauric acid at 70 degrees C, and the optimal conditions for this system have been defined. This first high-temperature in vitro catalytic system represents an important step in the development of industrially relevant catalysts.

Two notable features of the thermophilic CYP119, an Arg154-Glu212 salt bridge between the F-G loop and the I helix and an extended aromatic cluster, were studied to determine their contributions to the thermal stability of the enzyme. Site-specific mutants of the salt bridge (Arg154, Glu212) and aromatic cluster (Tyr2, Trp4, Trp231, Tyr250, Trp281) were expressed and purified. The substrate-binding and kinetic constants for lauric acid hydroxylation are little affected in most mutants, but the E212D mutant is inactive and the R154Q mutant has higher K(s),K(m), and k(cat) values. The salt bridge mutants, like wild-type CYP119, melt at 91+/-1 degrees C, whereas mutation of individual residues in the extended aromatic cluster lowers the T(m) by 10-15 degrees C even though no change is observed on mutation of an unrelated aromatic residue. The extended aromatic cluster, but not the Arg154-Glu212 salt bridge, contributes to the thermal stability of CYP119.