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.
Highlights
Nitric oxide (NO) is a small gaseous molecule, with a short half-life of a few seconds [1]
To gain insight into this new function of AHbs, we have focused on the role of distal histidine (HisE7) and its protonation states in modulating the protein nitrite reactivity in both Arabidopsis Hemoglobins 1 (AHb1) and AHb2 through the study of the AHb1 H69L and AHb2 H66L mutants
Consistent with the observed nitrite reductase rates, nitric oxide (NO) released from AHb2 H66L was significantly higher compared to AHb2 wt, while NO release was slightly decreased in AHb1 H69L compared to AHb1 wt
Summary
Nitric oxide (NO) is a small gaseous molecule, with a short half-life of a few seconds [1]. Nitrite reductase activity has been proposed for animals, cyanobacteria and non-symbiotic plant hemoglobin (nsHbs) based on their ability to reduce nitrite into NO [7,8,9,10]. This activity seems to be an intrinsic property of heme-containing globins. Class 2 nsHbs seem to be exclusive to dicots [20], and their gene expression is upregulated when plants experience low temperatures [15] They show tighter hexacoordination compared to class 1 nsHbs and have lower affinities for oxygen (Km « 100–200 nM) [21,22]. From animal and symbiotic Hbs, class 1 and class 2 nsHb are hexacoordinated in both the ferric and ferrous state; this is related to the presence of a histidine in the distal pocket, which binds the sixth coordination site of the heme iron in the absence of external ligands in a reversible manner [13]
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