CYP175A1 is a thermophilic cytochrome P450 and hydroxylares beta-carotene. We previously identified a native electron transport system for CYP175A1. In this report, We Constructed two fusion proteins consisting of CYP175A1, ferredoxin (Fdx), and ferredoxin-NADP(+) reductase (FNR): H2N-CYP175A1-Fdx-FNR-COOH (175FR)and H2N-CYP175A1-FNR-Fdx-COOH (175RF). Both 175FR and 175RF were expressed in Escherichia coli and purified. The V-max value for beta-carotene hydroxylation was 25 times higher with 175RF than 175FR and 9 times higher with 175RF than CYP175A1 (non-fused protein), although the k(m) values of these enzymes were similar. 175RF retained 50% residual activity even at 80 degrees C. Furthermore, several mutants of the CYP175A1 domain of 175RF were prepared and one mutant (Q67G/Y68I) catalyzed the hydroxylation of an unnatural substrate, testosterone. Thus. this is the first report of a thermostable Self-sufficient cytochrome P450 and the engineering of a thermophilic cytochrome P450 for the oxidation of an unnatural substrate. (c) 2009 Elsevier Inc. All rights reserved.