Flavin reductase: sequence of cDNA from bovine liver and tissue distribution.

Flavin reductase catalyzes electron transfer from reduced pyridine nucleotides to methylene blue or riboflavin, and this catalysis is the basis of the therapeutic use of methylene blue or riboflavin in the treatment of methemoglobinemia. A cDNA for a mammalian flavin reductase has been isolated and sequenced. Degenerate oligonucleotides, with sequences based on amino acid sequences of peptides derived from bovine erythrocyte flavin reductase, were used as primers in PCR to selectively amplify a partial cDNA that encodes the bovine reductase. The template used in the PCR was first strand cDNA synthesized from bovine liver total RNA using oligo(dT) primers. A PCR product was used as a specific probe to screen a bovine liver cDNA library. The sequence determined from two overlapping clones contains an open reading frame of 621 nucleotides and encodes 206 amino acids. The amino acid sequence deduced from the bovine liver flavin reductase cDNA matches the amino acid sequences determined for erythrocyte reductase-derived peptides, and the predicted molecular mass of 22,001 Da for the liver reductase agrees well with the molecular mass of 21,994 Da determined for the erythrocyte reductase by electrospray mass spectrometry. The amino acid sequence at the N terminus of the reductase has homology to sequences of pyridine nucleotide-dependent enzymes, and the predicted secondary structure, beta alpha beta, resembles the common nucleotide-binding structural motif. RNA blot analysis indicates a single 1-kilobase reductase transcript in human heart, kidney, liver, lung, pancreas, placenta, and skeletal muscle.

Pyrroloquinoline quinone acts with flavin reductase to reduce ferryl myoglobin in vitro and protects isolated heart from re-oxygenation injury.

Pyrroloquinoline quinone has been isolated from bacteria and recently has been detected in mammalian tissues and fluids. We report in vitro studies which show that pyrroloquinoline quinone serves as a high-affinity substrate for an erythrocyte "flavin reductase" and that the pyrroloquinoline quinol generated by this catalysis reacts rapidly with ferryl myoglobin radical. Western blot analysis of rat and rabbit heart homogenates detects a cross-reactive protein which has a molecular weight identical to the erythrocyte reductase from the same species. Low concentrations of pyrroloquinoline quinone protect isolated rabbit heart from re-oxygenation injury, serving as an effective tissue-protective agent in this model for cellular oxidative damage. We propose that this tissue protection is due to a pyrroloquinoline quinol-mediated reduction of reactive oxygen species.

Evidence that NADPH-dependent methemoglobin reductase and administered riboflavin protect tissues from oxidative injury.

NADPH-dependent methemoglobin reductase, first detected in erythrocytes sixty years ago, has subsequently been purified and characterized as a methylene blue reductase and a flavin reductase. The reductase plays no role in methemoglobin reduction under normal conditions, but its activity serves as the basis for the treatment of methemoglobinemia with methylene blue or flavin. On-going studies demonstrate that this cytosolic protein is also present in liver and that its primary structure distinguishes it from other known proteins. The bovine erythrocyte reductase tightly binds hemes, porphyrins, and fatty acids with resulting loss of activity. Pyrroloquinoline quinone serves as a high-affinity substrate of the reductase, suggesting that this naturally-occurring compound may be a physiological substrate. The ability of the reductase to catalyze the intracellular reduction of administered riboflavin to dihydroriboflavin suggested that this system might be exploited to protect tissues from oxidative damage. This hypothesis was supported by our finding that dihydroriboflavin reacts rapidly with Fe(IV)O and Fe(V)O oxidation states of hemeproteins, states that have been implicated in tissue damage associated with ischemia and reperfusion. Preliminary studies demonstrate that, as predicted, administration of low concentrations of riboflavin protects isolated rabbit heart from reoxygenation injury, rat lung from injury resulting from systemic activation of complement, and rat brain from damage caused by four hours of ischemia. Data from these animal studies suggest that flavin therapy holds promise in protecting tissue from the oxidative injuries of myocardial infarction, acute lung injury, stroke, and a number of other clinical conditions.

Characterization of NADPH-dependent methemoglobin reductase as a heme-binding protein present in erythrocytes and liver.

An NADPH-dependent reductase, first shown in the 1930s to catalyze the methylene blue-dependent reduction of methemoglobin in erythrocytes, has now been characterized as a high-affinity heme-binding protein and has been detected in liver. Highly purified bovine erythrocyte reductase binds protohemin to form a 1:1 complex with a Kd of 7 nM. Binding of protohemin completely inhibits reductase activity. Other tetrapyrroles and fatty acids also bind to the reductase and inhibit its activity. Protoporphyrin, hematoporphyrin, and coproporphyrin form 1:1 complexes with Kd values ranging from 1 to 5 microM. The inhibition constants for a number of saturated and unsaturated fatty acids range from 6 to 52 microM. A protein that is immunologically cross-reactive to the reductase has been detected in the cytosolic fractions of bovine and rat liver and of bovine, rat, rabbit, and human erythrocytes. By immunoblot analysis, the bovine liver and erythrocyte proteins appear identical in size, as do the rat liver and erythrocyte proteins. The concentration of the protein in bovine erythrocytes has been estimated by quantitative immunoblotting to be 10 microM. The detection of this protein in liver cells, the demonstration of its binding properties, and its weak reductase activity bring into question the long-held belief that this is uniquely an erythrocyte protein and that it functions as a reductase.

Evidence that the protein components of bovine erythrocyte green heme binding protein and flavin reductase are identical.

Bovine erythrocyte green heme binding protein and bovine erythrocyte flavin reductase have been isolated in highly purified forms and subjected to amino acid analysis and N-terminal amino acid sequence analysis. The two proteins possess similar amino acid compositions and identical N-terminal amino acid sequences. Moreover, the two proteins are immunochemically cross-reactive and are indistinguishable when compared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by double diffusion technique. This study provides evidence that the protein components of bovine erythrocyte green heme binding protein and flavin reductase are identical.