title: Nitric oxide scavenging by barley hemoglobin is facilitated by a monodehydroascorbate reductase-mediated ascorbate reduction of methemoglobin.
abstract: NADH-dependent NO scavenging in barley extracts is linked to hemoglobin (Hb) expression and is inhibited by SH-reagents. Barley Hb has a single cysteine residue. To determine whether this cysteine was critical for NO scavenging, barley Hb and a mutated version, in which the single Cys(79) was replaced by Ser, were over-expressed in Escherichia coli and purified to near homogeneity. The purified proteins exhibited very low NO-scavenging activity (12-14 nmol min(-1) mg(-1) protein) in the presence of NADH or NADPH. This activity was insensitive to SH-reagents. Addition of an extract from barley roots to either of the purified proteins resulted in high NADH-dependent NO turnover in a reaction that was sensitive to SH-reagents. A protein was purified from barley roots and identified by mass-spectrometry analysis as a cytosolic monodehydroascorbate reductase. It efficiently supported NADH-dependent NO scavenging in the presence of either native or mutated barley Hb. Ascorbate strongly facilitated the rate of metHb reduction. The K (m) for Hb was 0.3 microM, for ascorbate 0.6 mM and for NADH 4 microM. The reaction in the presence of monodehydroascorbate reductase was sensitive to SH-reagents with either form of the Hb. We conclude that metHb reduction and NO turnover do not involve direct participation of the Cys(79) residue of barley Hb. NO scavenging is facilitated by monodehydroascorbate reductase mediating a coupled reaction involving ferric Hb reduction in the presence of ascorbate and NADH.
authors.affiliation: Department of Plant Science, University of Manitoba, Winnipeg, MB, R3T 2N2 Canada.
authors.forename: Abir U
authors.initials: AU
authors.lastname: Igamberdiev
authors.forename: Natalia V
authors.initials: NV
authors.lastname: Bykova
authors.forename: Robert D
authors.initials: RD
authors.lastname: Hill
mesh_terms: D001205:Ascorbic Acid / Q000378:metabolism; D003545:Cysteine; D006454:Hemoglobins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D001467:Hordeum / Q000737:chemistry / Q000201:enzymology / Q000378:metabolism*; D008706:Methemoglobin / Q000378:metabolism; D050505:Mutant Proteins / Q000302:isolation & purification; D009243:NAD / Q000378:metabolism; D009247:NADH, NADPH Oxidoreductases / Q000032:analysis / Q000378:metabolism*; D009569:Nitric Oxide / Q000378:metabolism*; D010940:Plant Proteins / Q000032:analysis / Q000378:metabolism; D011994:Recombinant Proteins / Q000302:isolation & purification
chemical_list: D006454:Hemoglobins; D050505:Mutant Proteins; D010940:Plant Proteins; D011994:Recombinant Proteins; D009243:NAD; D009569:Nitric Oxide; D008706:Methemoglobin; D009247:NADH, NADPH Oxidoreductases; C023927:monodehydroascorbate reductase (NADH); D003545:Cysteine; D001205:Ascorbic Acid