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Int. J. Mol. Sci. 2016, 17(5), 640; doi:10.3390/ijms17050640

Residues in the Distal Heme Pocket of Arabidopsis Non-Symbiotic Hemoglobins: Implication for Nitrite Reductase Activity

1
Hypertension & Vascular Research Division, Department of Internal Medicine, Henry Ford Hospital, Detroit, MI 48202, USA
2
Department of Biotechnology, University of Verona, Strada Le Grazie 15, 37134 Verona, Italy
These authors contributed equally to this work.
*
Author to whom correspondence should be addressed.
Academic Editor: Eva Freisinger
Received: 16 February 2016 / Revised: 13 April 2016 / Accepted: 19 April 2016 / Published: 28 April 2016
(This article belongs to the Section Biochemistry, Molecular and Cellular Biology)
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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. View Full-Text
Keywords: Arabidopsis hemoglobins; nitrite reduction; distal cavity; hypoxia Arabidopsis hemoglobins; nitrite reduction; distal cavity; hypoxia
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Kumar, N.; Astegno, A.; Chen, J.; Giorgetti, A.; Dominici, P. Residues in the Distal Heme Pocket of Arabidopsis Non-Symbiotic Hemoglobins: Implication for Nitrite Reductase Activity. Int. J. Mol. Sci. 2016, 17, 640.

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