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. 1996 Jul;5(7):1389–1393. doi: 10.1002/pro.5560050717

Probing the structure of the linker connecting the reductase and heme domains of cytochrome P450BM-3 using site-directed mutagenesis.

S Govindaraj 1, T L Poulos 1
PMCID: PMC2143464  PMID: 8819171

Abstract

Cytochrome P450BM-3 is a catalytically self-sufficient fatty acid hydroxylase containing one equivalent each of heme, FMN, and FAD. The heme and flavins reside in separate domains connected by a linker peptide. In an earlier study (Govindaraj S, Poulos T, 1995, Biochemistry 34:11221-11226), we found that the length but not the sequence of the linker connecting the heme and reductase domains is important for enzyme activity. In the present study, residues in the linker were replaced with Pro and Gly to probe the role that regular secondary structure plays in linker function. The rate of flavin-to-heme electron transfer and the fatty acid hydroxylase activities of the glycine and proline substitution mutants, including a six-proline substitution, did not change significantly relative to wild-type enzyme. These results indicate that the linker does not adopt any regular secondary structure essential for activity and that the length of the linker is the critical feature that controls flavin-to-heme electron transfer.

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Selected References

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  1. Bredt D. S., Snyder S. H. Isolation of nitric oxide synthetase, a calmodulin-requiring enzyme. Proc Natl Acad Sci U S A. 1990 Jan;87(2):682–685. doi: 10.1073/pnas.87.2.682. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Gonvindaraj S., Li H., Poulos T. L. Flavin supported fatty acid oxidation by the heme domain of Bacillus megaterium cytochrome P450BM-3. Biochem Biophys Res Commun. 1994 Sep 30;203(3):1745–1749. doi: 10.1006/bbrc.1994.2388. [DOI] [PubMed] [Google Scholar]
  3. Govindaraj S., Poulos T. L. Role of the linker region connecting the reductase and heme domains in cytochrome P450BM-3. Biochemistry. 1995 Sep 5;34(35):11221–11226. doi: 10.1021/bi00035a031. [DOI] [PubMed] [Google Scholar]
  4. Govindaraj S., Poulos T. L. Role of the linker region connecting the reductase and heme domains in cytochrome P450BM-3. Biochemistry. 1995 Sep 5;34(35):11221–11226. doi: 10.1021/bi00035a031. [DOI] [PubMed] [Google Scholar]
  5. Hevel J. M., White K. A., Marletta M. A. Purification of the inducible murine macrophage nitric oxide synthase. Identification as a flavoprotein. J Biol Chem. 1991 Dec 5;266(34):22789–22791. [PubMed] [Google Scholar]
  6. Kunkel T. A., Roberts J. D., Zakour R. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods Enzymol. 1987;154:367–382. doi: 10.1016/0076-6879(87)54085-x. [DOI] [PubMed] [Google Scholar]
  7. Narhi L. O., Fulco A. J. Characterization of a catalytically self-sufficient 119,000-dalton cytochrome P-450 monooxygenase induced by barbiturates in Bacillus megaterium. J Biol Chem. 1986 Jun 5;261(16):7160–7169. [PubMed] [Google Scholar]
  8. Narhi L. O., Fulco A. J. Identification and characterization of two functional domains in cytochrome P-450BM-3, a catalytically self-sufficient monooxygenase induced by barbiturates in Bacillus megaterium. J Biol Chem. 1987 May 15;262(14):6683–6690. [PubMed] [Google Scholar]
  9. O'Keeffe D. H., Ebel R. E., Peterson J. A. Purification of bacterial cytochrome P-450. Methods Enzymol. 1978;52:151–156. doi: 10.1016/s0076-6879(78)52017-x. [DOI] [PubMed] [Google Scholar]
  10. Robbins A. H., Stout C. D. Structure of activated aconitase: formation of the [4Fe-4S] cluster in the crystal. Proc Natl Acad Sci U S A. 1989 May;86(10):3639–3643. doi: 10.1073/pnas.86.10.3639. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Sevrioukova I. F., Peterson J. A. NADPH-P-450 reductase: structural and functional comparisons of the eukaryotic and prokaryotic isoforms. Biochimie. 1995;77(7-8):562–572. doi: 10.1016/0300-9084(96)88172-7. [DOI] [PubMed] [Google Scholar]
  13. Sharp R. E., White P., Chapman S. K., Reid G. A. Role of the interdomain hinge of flavocytochrome b2 in intra- and inter-protein electron transfer. Biochemistry. 1994 May 3;33(17):5115–5120. doi: 10.1021/bi00183a015. [DOI] [PubMed] [Google Scholar]
  14. WILD H. Uber periproktitische Abszesse. Wien Med Wochenschr. 1951 Mar 3;101(9):159–161. [PubMed] [Google Scholar]
  15. Xia Z. X., Mathews F. S. Molecular structure of flavocytochrome b2 at 2.4 A resolution. J Mol Biol. 1990 Apr 20;212(4):837–863. doi: 10.1016/0022-2836(90)90240-M. [DOI] [PubMed] [Google Scholar]
  16. Zhang M., Vogel H. J. Characterization of the calmodulin-binding domain of rat cerebellar nitric oxide synthase. J Biol Chem. 1994 Jan 14;269(2):981–985. [PubMed] [Google Scholar]

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