Leghemoglobin reductase

In enzymology, a leghemoglobin reductase (EC 1.6.2.6) is an enzyme that catalyzes the chemical reaction

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

The 4 substrates of this enzyme are NADH, NADPH, H⁺, and ferrileghemoglobin, whereas its 3 products are NAD⁺, NADP⁺, and ferroleghemoglobin.

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 of ferrous leghemoglobin in legume nodules.

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]

The discovery of ferric leghemoglobin reductases.

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^[9] 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 and other legume ferric leghemoglobin reductases.

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 K_m and K_cat values of soybean FLbR for soybean Lb³⁺ reduction are 9.2 μM and 6.2 s⁻¹, respectively (K_cat/K_m = 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 K_m and K_cat values of cowpea FLbR for cowpea Lb³⁺ reduction are 10.4 μM and 3.1 s⁻¹, respectively (K_cat/K_m = 298 M⁻¹ s⁻¹).^[13]

Ferric leghemoglobin reductases as possible reductants of phytoglobins in vivo.

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⁺. 

References

• Saari LL, Klucas RV (1984). "Ferric leghemoglobin reductase from soybean root nodules". Arch. Biochem. Biophys. 231 (1): 102–13. PMID 6539095. doi:10.1016/0003-9861(84)90367-9.