NADH-dependent metabolism of nitric oxide in alfalfa root cultures expressing barley hemoglobin

Abstract

Transgenic alfalfa ( Medicago sativa L.) root cultures expressing sense and antisense barley ( Hordeum vulgare L.) hemoglobin were examined for their ability to metabolize NO. Extracts from lines overexpressing hemoglobin had approximately twice the NO conversion rate of either control or antisense lines under normoxic conditions. Only the control line showed a significant increase in the rate of NO degradation when placed under anaerobic conditions. The decline in NO was dependent on the presence of reduced pyridine nucleotide, with the NADH-dependent rate being about 2.5 times faster than the NADPH-dependent rate. Most of the activity was found in the cytosolic fraction of the extracts, while only small amounts were found in the cell wall, mitochondria, and 105,000- g membrane fraction. The NADH-dependent NO conversion exhibited a broad pH optimum in the range 7-8 and a strong affinity to NADH and NADPH ( K(m) 3 microM for both). It was sensitive to diphenylene iodonium, an inhibitor of flavoproteins. The activity was strongly reduced by applying antibodies raised against recombinant barley hemoglobin. Extracts of Escherichia coli overexpressing barley hemoglobin showed a 4-fold higher rate of NO metabolism as compared to non-transformed cells. The NADH/NAD and NADPH/NADP ratios were higher in lines underexpressing hemoglobin, indicating that the presence of hemoglobin has an effect on these ratios. They were increased under hypoxia and antimycin A treatment. Alfalfa root extracts exhibited methemoglobin reductase activity, using either cytochrome c or recombinant barley hemoglobin as substrates. There was a correspondence between NO degradation and nitrate formation. The activity was eluted from a Superose 12 column as a single peak with molecular weight of 35+/-4 kDa, which corresponds to the size of the hemoglobin dimer. The results are consistent with an NO dioxygenase-like activity, with hemoglobin acting in concert with a flavoprotein, to metabolize NO to nitrate utilizing NADH as the electron donor.

Arabidopsis Nonsymbiotic Hemoglobin AHb1 Modulates Nitric Oxide Bioactivity

“…These observations, corroborated by the direct correlation between weight of shoots and AHb1 level observed after exposure of wild-type and transgenic Arabidopsis plants to hypoxic stress (Figure 6) and by the large accumulation of NO observed in hypoxic root cultures of hemoglobin-deficient alfalfa (Dordas et al, 2003b) and maize (Zea mays) mutants (Dordas et al, 2004), support the hypothesis that stress-induced hemoglobins may function as NO dioxygenases (Dordas et al, 2003a;Igamberdiev et al, 2004) protecting against nitrosative stress associated with hypoxia and confirm that AHb1 metabolizes NO in vivo.…”

“…In the presence of saturating concentrations of NO, the specific activity of AHb1 was 24 nmol/ min/mg. Because methemoglobin reductase activity was found in alfalfa (Medicago sativa) root cultures (Igamberdiev et al, 2004), it is possible that AHb1 activity may be augmented in vivo by specific interaction with a reductase system, as suggested for barley (Hordeum vulgare) hemoglobin (Igamberdiev et al, 2004) and demonstrated for human myoglobin (Flogel et al, 2001).…”

Nitric oxide accelerates seed germination in warm-season grasses

“…Among potential cellular targets for CN -or NO would be hemoproteins, such as cytochromes, hemoglobins, heme-containing reductases and heme-containing oxidases (e.g., Kundu et al 2003;Igamberdiev et al 2004). NO, in addition can S-nitrosylate plant proteins (Lindemayr et al 2005).…”

Oxygen and nitric oxide rebinding kinetics in nonsymbiotic hemoglobin AHb1 from Arabidopsis thaliana

“…The very high oxygen affinity, due to a very low dissociation rate constant, means that AHb1 strongly stabilizes O 2 after binding, thus ruling out a possible function related to O 2 sensing, carrying, or storing (6), whereas a role in electron transport is unlikely because of its high redox potential (7,8). As suggested in general for class 1 nsHbs (9), and proved in vivo for maize and alfalfa lines overexpressing class 1 nsHbs (8,10), AHb1 may participate in NO detoxification by acting as NO scavenger under hypoxic stress. In fact, the rapid nitrate accumulation from the reaction of oxyAHb1 with NO in vitro may be physiologically relevant in vivo to reduce the levels of NO under hypoxic stress (11,12).…”