In enzymology, a leghemoglobin reductase () is an enzyme that catalyzes the chemical reaction

NAD(P)H + H+ + 2 ferrileghemoglobin NAD(P)+ + 2 ferroleghemoglobin

In other words, a leghemoglobin (or phytoglobin in general) with a Fe3+ is reduced to one with the ferrous ion, Fe2+.

This enzyme belongs to the family of oxidoreductases, specifically those acting on NADH or NADPH with a heme protein as acceptor. The systematic name of this enzyme class is NAD(P)H:ferrileghemoglobin oxidoreductase. This enzyme is also called ferric leghemoglobin reductase.

Role in legume nodules

Leghemoglobin (Lb) is a heme-containing protein that reversibly binds and transports O2 into the N2-fixing nodules of leguminous plants.Appleby C. A., The origin and functions of haemoglobin in plants, Sci. Progress, 76 (1992) 365-398. In order to function as an O2-carrier Lb must be in the ferrous oxidation state (Lb2+). Oxygenated Lb2+ (Lb2+O2) readily autoxidizes to ferric Lb (Lb3+) generating O2− in the presence of trace amounts of transition metals, chelators and toxic metabolites (such as nitrite, superoxide radical and peroxides),Becana M., Klucas R. V., Oxidation and reduction of leghemoglobin in root nodules of leguminous plants, Plant Physiol., 98 (1992) 1217-1221. however Lb2+ is the predominant form in nodules.Lee K. K., Klucas R. V., Reduction of ferric leghemoglobin in soybean root nodules, Plant Physiol., 74 (1984) 984-988.Lee K. K., Shearman L. L., Ericson B. K., Klucas R. V., Ferric leghemoglobin in plant-attached leguminous nodules., Plant Physiol., 109 (1995) 261-267. Therefore, mechanisms exist in vivo for maintaining Lb in the functional ferrous status.Becana M., Klucas R. V., Enzymatic and nonenzymatic mechanisms for ferric leghemoglobin reduction in legume root nodules, Proc. Natl. Acad. Sci. USA, 87 (1990) 7295-7299.

History

Burris and HassBurris R. H., Hass E., The red pigment of leguminous root nodules., J. Biol. Chem., 155 (1944) 227-229. were the first to propose that reduced pyridine nucleotides might function as reductants of Lb3+ in leguminous root nodules and in 1969 ApplebyAppleby C. A., Properties of leghaemoglobin in vivo, and its isolation as ferrous oxyleghaemoglobin, Biochim Biophys. Acta, 188 (1969) 222-229. reported that Lb3+ was reduced to Lb2+ by a suspension of bacteroids. In 1982 Kretovich and collaboratorsKretovich V. L., Melik-Sarkisyan S. S., Bashirova N. F., Topunov A. F., Enzymatic reduction of leghemoglobin in lupin nodules, J. Appl. Biochem., 4 (1982) 209-217. purified an enzyme from lupine nodules which catalyzed the reduction of Lb3+ to Lb2+ using NADH as reductant. This enzyme (named by these authors as Legoglobin Reductase -LR) is similar to NADH:cytochrome b5 reductase (EC 1.6.2.2) from erythrocytes and bovine muscle. Lupin LR is a flavoprotein with a molecular mass of 60 kDa and its activity is specific for NADH. In 1984 Klucas and collaboratorsSaari L. L., Klucas R. V., Ferric leghemoglobin reductase from soybean root nodules, Arch. Biochem. Biophys., 231 (1984) 102-113. purified a protein with ferric Lb reductase (FLbR) activity from soybean nodules. The activity of soybean FLbR was 90% in the nodule cytosol and 10% in the bacteroids. NADH was the best reductant for soybean FLbR, although NADPH also functioned at rates that were three-fold less than NADH. These investigations by Klucas and collaborators also showed that the oxidation of NADH and reduction of Lb3+ was undetectable when O2 was removed from the reaction system, but all were restored upon re-addition of O2, which indicated that the FLbR activity is O2-dependent.

In legumes

Soybean FLbR is a flavoprotein with flavin adenine dinucleotide (FAD) as the prosthetic group and consists of two identical subunits, each having a molecular mass of 54 kDa. The Km and Kcat values of soybean FLbR for soybean Lb3+ reduction are 9.2 μM and 6.2 s−1, respectively (Kcat/Km = 674 M−1 s−1). The amino acid sequence of soybean FLbR is highly related to that of the flavin-nucleotide disulfide oxidoreductases, especially dihydrolipoamide dehydrogenase (DLDH) (EC 1.8.1.4) of the pyruvate dehydrogenase complex. The amino acid sequence of soybean FLbR contains a 30-residue signal peptide for translocation into the mitochondria as well as conserved regions for the FAD-binding site, NAD(P)H-binding site and disulfide active site characteristic of pea DLDH and other enzymes in the family of the pyridine nucleotide-disulfide oxidoreductases.Ji L., Wood S., Becana M., Klucas R. V., Purification and characterization of soybean root nodule ferric leghemoglobin reductase, Plant Physiol., 96 (1991) 32-37.

The soybean genome contains at least two copies (named flbr1 and flbr2) of the flbr gene.Ji L., Becana M., Sarath G., Klucas R. V., Cloning and sequence analysis of a cDNA encoding ferric leghemoglobin reductase from soybean nodules, Plant Physiol., 104 (1994) 453-459. The amino acid sequence of soybean FLbR2 has considerable homology with soybean FLbR1 and pea leaf mitochondria DLDH and contains a 30-residue mitochondrial transit peptide.Moran J. F., Sun Z., Sarath G., Arredondo-Peter R., James E. K., Becana M., Klucas R. V., Molecular cloning, functional characterization, and subcellular localization of soybean nodule dihydrolipoamide reductase., Plant Physiol., 128 (2002) 300-313. FLbR sequences have also been detected and analyzed in legumes other than soybean. For example, the nucleotide sequence of a cowpea FLbR cDNA has 88 and 85% similarity with soybean FLbR and pea DLDH, respectively. The Km and Kcat values of cowpea FLbR for cowpea Lb3+ reduction are 10.4 μM and 3.1 s−1, respectively (Kcat/Km = 298 M−1 s−1).Luan P., Aréchaga-Ocampo E., Sarath G., Arredondo-Peter R., Klucas R. V., Analysis of a ferric leghemoglobin reductase from cowpea (Vigna unguiculata) root nodules., Plant Sci., 154 (2000) 161-170.

In other plants

Soybean FLbR2 reduces ferric rice Phytoglobin1.1 (Phytogb1.13+).Gopalasubramaniam S. K., Kondapalli K. C., Millán-Pacheco C., Pastor N., Stemmler T. L., Moran J. F., Arredondo-Peter R., Soybean dihydrolipoamide dehydrogenase (ferric leghemoglobin reductase 2) interacts with and reduces ferric non-symbiotic hemoglobin 1., ScienceJet, 2 (2013) 33. Apparently, the soybean FLbR2-rice Phytoglobin1.13+ interaction is weak. An in silico analysis predicted that soybean FLbR2 and rice Phytogb1.13+ interact at the FAD-binding domain of soybean FLbR2 and the CD-loop and helix F of rice Phytogb1.13+. Therefore, FLbRs could be a generalized in vivo mechanism for the enzymatic reduction of Phytogbs3+.

References

See also Cytochrome b5 reductase