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Probing the Mechanisms of the Biological Intermolecular Transfer of Reduced Flavin^{1 ,2}

Department of Biology and Biochemistry, University of Houston, Houston, TX 77204-5513

³To whom correspondence should be addressed.

	ABSTRACT

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NAD(P)H-flavin oxidoreductases [flavin reductases (FR)] are a class of enzymes capable of producing reduced flavin for bacterial bioluminescence and other biological processes. Bacterial luciferase utilizes oxygen, reduced FMN (FMNH₂) and a long-chain aliphatic aldehyde as substrates for light emission. The Vibrio harveyi luciferase and FRP (for which we have cloned the gene and determined the crystal structure) is a model for the elucidation of the reduced flavin transfer mechanism using both a flavin reductase single-enzyme assay monitoring the NADPH oxidation and a flavin reductase-luciferase coupled assay measuring bioluminescence intensity or quantum output. The FRP exhibits a ping-pong kinetic pattern in the single-enzyme assay but changes to a sequential pattern in the coupled assay. Furthermore, FMN at >2 x10^-6 mol/L reduced both the light intensity and quantum yield of the coupled reaction by noncompetitively inhibiting NADPH and competitively inhibiting luciferase. These results support a scheme in which the luciferase forms specific complex(es) with FRP. Indeed, such complexes were shown by fluorescence anisotropy to exist between luciferase and monomeric FRP either in the holo- or apoenzyme form. Furthermore, the reduced flavin cofactor of FRP is transferred directly to luciferase for bioluminescence, whereas the reduced flavin product of FRP is inefficient in supporting the luminescence reaction. The mechanism of reduced flavin transfer is apparently flavin and flavin reductase specific.

KEY WORDS: • flavin reductases • luciferase, bacterial • flavin, reduced • transfer, reduced flavin • channeling, reduced flavin

	INTRODUCTION

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An enzyme acquires its specific substrate in vivo either through binding of the substrate, which reaches the enzyme active site by diffusion, or through channeling of such a substrate from a specific donor in a stable or transient complex with the acceptor enzyme. Mechanisms of the latter direct transfer processes have attracted considerable research interest. Reduced flavins are subject to rapid autoxidation, with a lifetime of <1 s in an air-saturated aqueous medium. Consequently, free diffusion cannot be an efficient way of transfer, and specific channeling is expected to exist for at least some donor-acceptor pairs. However, the nature of reduced flavin transfer in biological systems remains essentially unexplored at present.

NAD(P)H-flavin oxidoreductases (flavin reductases) are a class of enzymes capable of producing reduced flavin using NAD(P)H as a reductant. An increasing number of biological processes have been found to require exogenous reduced flavin for their functions. Flavin reductases (FR) are either effective in activating or essential to these processes such as bacterial bioluminescence, reduction of methemoglobin and ferrylmyoglobin, reductive iron release from siderophores, oxygen activation, activation of ribonucleotide reductase and chorismate synthase, biosynthesis of the antitumor agent valanimycin and desulfurization of fossil fuel [see Lei and Tu (1998), and references therein]. The bacterial luciferase utilizes oxygen, reduced FMN (FMNH₂) and a long-chain aliphatic aldehyde as substrates for light emission (Hastings and Nealson 1977 ). The required FMNH₂ is believed to be supplied in vivo by at least one species of the three types of flavin reductases (namely, the NADH-preferring FRD, the NADPH-preferring FRP and the general FRG, which utilizes both NADH and NADPH) known to exist in luminous bacteria (Duane and Hastings 1975 , Gerlo and Charlier 1975 , Jablonski and DeLuca 1978 , Watanabe and Hastings 1982 ). We have cloned the gene encoding the Vibrio harveyi FRP (Lei et al. 1994 ) and determined its crystal structure (Tanner et al. 1996 ). FRP is a homodimer with each monomer (26,000 MW) having one tightly bound FMN cofactor (Lei et al. 1994 ). We have chosen the V. harveyi luciferase and FRP as a model for the elucidation of the reduced flavin transfer mechanism.

The kinetic mechanism of FRP was examined by using both the flavin reductase single-enzyme assay monitoring the NADPH oxidation and the flavin reductase-luciferase coupled assay measuring bioluminescence intensity or quantum output. The FRP exhibited a ping-pong kinetic pattern with a K_m, FMN of 8 x 10^-6 mol/L and a K_m, NADPH of 20 x 10^-6mol/L in the single-enzyme assay, but changed to a sequential pattern with a K_m, FMN of 0.3 x 10^-6 mol/L and a K_m, NADPH of 0.02 x 10^-6 mol/L in the coupled assay. Furthermore, FMN at >2 x 10^-6 mol/L reduced both the light intensity and quantum yield of the coupled reaction by noncompetitively inhibiting NADPH and competitively inhibiting luciferase. These results support a scheme in which the luciferase forms specific complex(es) with FRP. Indeed, such complexes were shown by fluorescence anisotropy to exist between luciferase and monomeric FRP either in the holo- or apoenzyme form. Furthermore, the reduced flavin cofactor of FRP is transferred directly to luciferase for bioluminescence, whereas the reduced flavin product of FRP is inefficient in supporting the luminescence reaction. When the FMN cofactor of FRP was replaced by 2-thioFMN, the resulting flavin reductase was highly active in reducing the FMN substrate in the single-enzyme assay but quite inefficient in the coupled assay for bioluminescence. Because reduced 2-thioFMN is known to be a poor substrate for luciferase (Mitchell and Hastings 1969 ), such a finding strongly supports the direct transfer of reduced flavin cofactor from FRP to luciferase. The mechanism of reduced flavin transfer is apparently flavin reductase specific. Using the Photobacterium fischeri FRG (which also has a tightly bound FMN cofactor) in a similar study, we found that the reduced flavin product rather than the cofactor was transferred preferentially to luciferase.

	FOOTNOTES

 
¹ Presented at the symposium entitled "Mechanistic Aspects of Vitamin and Coenzyme Utilization and Function: A Symposium in Recognition of the Distinguished Career of Donald B. McCormick" as part of the Experimental Biology 99 meeting held April 17–21 in Washington, DC. This symposium was sponsored by the American Society for Nutritional Sciences. The proceedings of this symposium are published as a supplement to The Journal of Nutrition. Guest editors for this supplement publication were Alfred H. Merrill, Jr., Emory University School of Medicine, Atlanta, GA, Barbara Bowman, U.S. Centers for Disease Control and Prevention, Atlanta, GA, and Peter C, Preusch, National Institutes of General Medical Sciences, Bethesda, MD.

² Supported by grants GM25953 from the National Institutes of Health and E-1030 from The Robert A. Welch Foundation.

	REFERENCES

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1. Duane W., Hastings J. W. Flavin mononucleotide reductase of luminous bacteria. Mol. Cell. Biochem. 1975;6:53-64[Medline]

2. Hastings J. W., Nealson K. H. Bacterial bioluminescence. Annu. Rev. Microbiol. 1977;31:549-595[Medline]

3. Gerlo E., Charlier J. Identification of NADH-specific and NADPH-specific FMN reductases in Beneckea harveyi. Eur. J. Biochem. 1975;57:461-467[Medline]

4. Jablonski E., DeLuca M. Studies of the control of luminescence in Beneckea harveyi: properties of the NADH and NADPH:FMN oxidoreductases. Biochemistry 1978;17:672-678[Medline]

5. Lei B., Liu M., Huang S., Tu S.-C. Vibrio harveyi NADPH:flavin oxidoreductase: cloning, sequence and overexpression of the gene and purification and characterization of the cloned enzyme. J. Bacteriol. 1994;176:3552-3558[Abstract/Free Full Text]

6. Lei B., Tu S.-C. Mechanism of reduced flavin transfer from Vibrio harveyi NADPH- FMN oxidoreductase to luciferase. Biochemistry 1998;37:14623-14629[Medline]

7. Mitchell G., Hastings J. W. The effect of flavin isomers and analogues upon the color of bacterial bioluminescence. J. Biol. Chem. 1969;244:2572-2576[Abstract/Free Full Text]

8. Tanner J. J., Lei B., Tu S.-C., Krause K. L. Flavin reductase P: structure of a dimeric enzyme that reduces flavin. Biochemistry 1996;35:13531-13539[Medline]

9. Watanabe H., Hastings J. W. Specificities and properties of three reduced pyridine nucleotide-flavin mononucleotide reductases coupling to bacterial luciferase. Mol. Cell. Biochem. 1982;44:181-187[Medline]

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