title: Residues in the Distal Heme Pocket of Arabidopsis Non-Symbiotic Hemoglobins: Implication for Nitrite Reductase Activity.
abstract: It is well-established that plant hemoglobins (Hbs) are involved in nitric oxide (NO) metabolism via NO dioxygenase and/or nitrite reductase activity. The ferrous-deoxy Arabidopsis Hb1 and Hb2 (AHb1 and AHb2) have been shown to reduce nitrite to NO under hypoxia. Here, to test the hypothesis that a six- to five-coordinate heme iron transition might mediate the control of the nitrite reduction rate, we examined distal pocket mutants of AHb1 and AHb2 for nitrite reductase activity, NO production and spectroscopic features. Absorption spectra of AHbs distal histidine mutants showed that AHb1 mutant (H69L) is a stable pentacoordinate high-spin species in both ferrous and ferric states, whereas heme iron in AHb2 mutant (H66L) is hexacoordinated low-spin with Lys69 as the sixth ligand. The bimolecular rate constants for nitrite reduction to NO were 13.3 ± 0.40, 7.3 ± 0.5, 10.6 ± 0.8 and 171.90 ± 9.00 M(-1)·s(-1) for AHb1, AHb2, AHb1 H69L and AHb2 H66L, respectively, at pH 7.4 and 25 °C. Consistent with the reductase activity, the amount of NO detected by chemiluminescence was significantly higher in the AHb2 H66L mutant. Our data indicate that nitrite reductase activity is determined not only by heme coordination, but also by a unique distal heme pocket in each AHb.
authors.affiliation: Hypertension &amp; Vascular Research Division, Department of Internal Medicine, Henry Ford Hospital, Detroit, MI 48202, USA. nkumar3@hfhs.org.|Department of Biotechnology, University of Verona, Strada Le Grazie 15, 37134 Verona, Italy. nkumar3@hfhs.org.
authors.forename: Nitin
authors.initials: N
authors.lastname: Kumar
authors.affiliation: Department of Biotechnology, University of Verona, Strada Le Grazie 15, 37134 Verona, Italy. alessandra.astegno@univr.it.
authors.forename: Alessandra
authors.initials: A
authors.lastname: Astegno
authors.affiliation: Department of Biotechnology, University of Verona, Strada Le Grazie 15, 37134 Verona, Italy. jianchen0722@gmail.com.
authors.forename: Jian
authors.initials: J
authors.lastname: Chen
authors.affiliation: Department of Biotechnology, University of Verona, Strada Le Grazie 15, 37134 Verona, Italy. alejandro.giorgetti@univr.it.
authors.forename: Alejandro
authors.initials: A
authors.lastname: Giorgetti
authors.affiliation: Department of Biotechnology, University of Verona, Strada Le Grazie 15, 37134 Verona, Italy. paola.dominici@univr.it.
authors.forename: Paola
authors.initials: P
authors.lastname: Dominici
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D017360:Arabidopsis / Q000378:metabolism*; D029681:Arabidopsis Proteins / Q000737:chemistry / Q000378:metabolism*; D006418:Heme / Q000737:chemistry / Q000378:metabolism; D006454:Hemoglobins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006861:Hydrogen Peroxide / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D008163:Luminescent Measurements; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009569:Nitric Oxide / Q000032:analysis / Q000378:metabolism; D009572:Nitrite Reductases / Q000737:chemistry / Q000378:metabolism*; D010084:Oxidation-Reduction; D030421:Peroxynitrous Acid / Q000737:chemistry; D016415:Sequence Alignment
chemical_list: D029681:Arabidopsis Proteins; D006454:Hemoglobins; D030421:Peroxynitrous Acid; D009569:Nitric Oxide; D006418:Heme; D006861:Hydrogen Peroxide; D009572:Nitrite Reductases
keywords: Arabidopsis hemoglobins; distal cavity; hypoxia; nitrite reduction