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Nitric oxide scavenging by barley hemoglobin is facilitated by a monodehydroascorbate reductase-mediated ascorbate reduction of methemoglobin

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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 Cys79 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 μM, for ascorbate 0.6 mM and for NADH 4 μM. 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 Cys79 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.

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Abbreviations

DEANO:

2-(N, N-diethylamino)-diazenolate-2-oxide

DTT:

Dithiothreitol

metHb:

Methemoglobin (ferric hemoglobin)

rHb:

Recombinant non-mutated barley Hb

mutHb:

Recombinant barley Hb with replacement of Cys79 by Ser

MDHA:

Monodehydroascorbate (ascorbate free radical)

MDHAR:

Monodehydroascorbate reductase

NEM:

N-ethylmaleimide

pHMB:

p-Hydroxymercuribenzoate

SNP:

Sodium nitroprusside

TCEP:

Tris(2-carboxyethyl)phosphine

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Acknowledgements

The skilful technical assistance of Doug Durnin is gratefully acknowledged. We thank Professor Werner Ens for mass spectrometry support. This work was supported by the Natural Sciences and Engineering Research Council of Canada (OGP 4689).

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Authors and Affiliations

  1. Department of Plant Science, University of Manitoba, Winnipeg, MB, R3T 2N2, Canada

    Abir U. Igamberdiev & Robert D. Hill

  2. Cereal Research Centre, Agriculture and Agri-Food Canada, Winnipeg, MB, R3T 2M9, Canada

    Natalia V. Bykova

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  1. Abir U. Igamberdiev
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  2. Natalia V. Bykova
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  3. Robert D. Hill
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Correspondence to Robert D. Hill.

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Igamberdiev, A.U., Bykova, N.V. & Hill, R.D. Nitric oxide scavenging by barley hemoglobin is facilitated by a monodehydroascorbate reductase-mediated ascorbate reduction of methemoglobin. Planta 223, 1033–1040 (2006). https://doi.org/10.1007/s00425-005-0146-3

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