| leghemoglobin reductase | |
| Ec Number: | 1.6.2.6 |
| Cas Number: | 60440-35-9 |
| Go Code: | 0015043 |
In enzymology, a leghemoglobin reductase is an enzyme that catalyzes the chemical reaction
NAD(P)H + H+ + 2 ferrileghemoglobin
\rightleftharpoons
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.
Leghemoglobin (Lb) is a heme-containing protein that reversibly binds and transports O2 into the N2-fixing nodules of leguminous plants.[1] 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),[2] however Lb2+ is the predominant form in nodules.[3] [4] Therefore, mechanisms exist in vivo for maintaining Lb in the functional ferrous status.[5]
Burris and Hass[6] were the first to propose that reduced pyridine nucleotides might function as reductants of Lb3+ in leguminous root nodules and in 1969 Appleby[7] reported that Lb3+ was reduced to Lb2+ by a suspension of bacteroids. In 1982 Kretovich and collaborators[8] 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 collaborators[9] 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.
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.[10]
The soybean genome contains at least two copies (named flbr1 and flbr2) of the flbr gene.[11] 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.[12] 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).[13]
See also: phytoglobin.
Soybean FLbR2 reduces ferric rice Phytoglobin1.1 (Phytogb1.13+).[14] 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+.