A wide range of biochemical functions are linked to the actions of monooxygenases which activate the triplet molecular oxygen for the insertion of one oxygen atom into singlet organic substrates. Flavoprotein hydroxylases, a sub- family of monooxygenases, are important in detoxification, drug activation or inactivation, biodegradation of naturally occurring and man-made organic compounds, bioluminescence, and other processes. The long term goal of this project is to acquire an integrated understanding of the structures and mechanisms of flavoprotein hydroxylases. During the past funding periods, research efforts have been directed toward selected flavoprotein aromatic hydroxylases and bacterial luciferase. For this application, the specific aims are to continue the structural and mechanistic delineations of bacterial luciferase, and to carry out enzymological and molecular biological investigations of flavin reductases as a new endeavor. Luciferase shares many common mechanistic properties with other flavohydroxylases. On the other hand, due to its extremely slow turnover and the unique ability to emit light, luciferase also offers special challenges and advantages for enzymological investigations. Moreover, luciferase and its genes provide one of the most versatile reporter systems for basic biomedical and biotechnological research. For the next funding period, both enzymatic and flavin model studies will be carried out to elucidate the mechanisms of the normal luminescence reaction catalyzed by luciferase and the spectrally altered luminescence induced by the coexistence of a lumazine protein or yellow fluorescent protein with luciferase. The successful site-directed mutagenesis studies on luciferase will be continued to investigate further the specific roles of the essential alpha H44 and alpha H45. Plans are also specified for resolving the current controversy over the reported activities of luciferase individual subunits. Unlike other flavohydroxylases, luciferase is unable to reduce flavin by NAD(P)H. The necessary FMNH2 substrate for luciferase is believed to be supplied in vivo by flavin reductases. Both earlier and recent kinetic results suggest a direct FMNH2 transfer from flavin reductases to luciferase. Relatively little is known about flavin reductases from luminous bacteria or other sources. The mechanism of an NADPH-specific flavin reductase (FRP) will be elucidated by equilibrium binding, state-state analysis, stopped-flow measurements, and isotope kinetic effects. The nature of FMNH2 transfer from FRP to luciferase and the likely complex formation between the two enzymes will be investigated by kinetic and fluorescence studies. The functional contribution of FRP to the in vivo bioluminescence will be examined by a gene knockout study. Similar investigations with a more limited scope will be extended to two other flavin reductase species. These studies will not only contribute to a better understanding of the flavin- pyridine nucleotide redox biochemistry but also explore the functional links between bioenergetics and bioluminescence.
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