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Volume 272, Number 36, Issue of September 5, 1997 pp. 22509-22513
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.

Negatively Charged Anabaena Flavodoxin Residues (Asp¹⁴⁴ and Glu¹⁴⁵) Are Important for Reconstitution of Cytochrome P450 17-Hydroxylase Activity

(Received for publication, May 27, 1997, and in revised form, July 1, 1997)

Christopher M. Jenkins , Carlos G. Genzor ^¶ , María F. Fillat ^¶ , Michael R. Waterman and Carlos Gómez-Moreno ^¶

From the  Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146 and ^¶ Departmento de Bioquímica y Biología Molecular y Celular, Facultad de Ciencias, Universidad de Zaragoza, 50009 Zaragoza Spain

Catalysis by microsomal cytochromes P450 requires the membrane-bound enzyme NADPH-cytochrome P450 reductase (P450 reductase), which transfers electrons to the P450 heme via a flavodoxin-like domain. Previously, we reported that Escherichia coli flavodoxin (Fld), a soluble electron transfer protein, directly interacts with bovine cytochrome P450 17-hydroxylase/17,20-lyase (P450c17) and donates electrons to this enzyme when reconstituted with NADPH-ferredoxin (flavodoxin) reductase (FNR) (Jenkins, C. M., and Waterman, M. R. (1994) J. Biol. Chem. 269, 27401-27408). To investigate whether flavodoxins can serve as useful models of the analogous domain in P450 reductase, we have examined the FNR-Fld system from the cyanobacterium Anabaena. Mutagenesis of two acidic Anabaena Fld residues (D144A and E145A) significantly decreased flavodoxin-supported P450c17 progesterone 17-hydroxylase activity. Specifically, D144A exhibited only 15% of the activity of wild-type Fld, whereas the adjacent mutation, E145A, caused a 40% loss in activity. P450-dependent hydrogen peroxide/superoxide production by wild-type FNR-Fld was measurably higher than that generated by FNR-D144A or FNR-E145A, indicating that the mutations do not lead to P450 heme-mediated electron uncoupling. Interestingly, the D144A and E145A mutants bind with equal or even greater affinity to P450c17 than wild-type Fld. Furthermore, these mutations (D144A and E145A) actually increased cytochrome c reductase activity (35 and 100% higher than wild type). Anabaena Fld residues Asp¹⁴⁴ and Glu¹⁴⁵ align closely with rat P450 reductase residue Asp²⁰⁸, which has been shown by mutagenesis to be important in electron transfer to P4502B1 but not to cytochrome c (Shen, A. L., and Kasper, C. B. (1995) J. Biol. Chem. 270, 27475-27480). Thus, these residues in flavodoxins and P450 reductase appear to have similar functions in P450 recognition and/or electron transfer, supporting the hypothesis that flavodoxins represent valid models for the FMN-binding domain of P450 reductase.

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