Structural and functional investigation of Arabidopsis thaliana non-symbiotic hemoglobins

Non-symbiotic plant hemoglobins (nsHbs) have been found in various plant tissues and plant species, especially in crop plants. Different classes of nsHbs have been identified and divided into class-1 (Hb1) and class-2 (Hb2) based on phylogenetic characteristics, gene expression pattern and oxygen binding properties. Differently from symbiotic hemoglobins, class 1 and class 2 nsHb are hexacoordinated in both ferric and ferrous state, due to a histidine in the distal pocket that reversibly binds the sixth coordination site of the heme iron, in the absence of external ligands. Class 1 non-symbiotic hemoglobins have a very high oxygen affinity (Km in the order of 2 nM) and their expression is highly induced by hypoxic stress. Low concentrations in vivo along with small oxygen dissociation rate constants indicate that these nsHbs are unlikely to function in oxygen transport, while high oxygen affinity and redox potential indicate they are unlikely to function in electron transport. Studies using Arabidopsis, maize and alfalfa lines over-expressing Hb1 indicate a role of class-1 Hbs in scavenging nitric oxide (NO) that is produced under severe hypoxia. Relatively little is known about the function of Hb2 in plants. Class 2 nsHbs seem to be exclusive to dicots and are overexpressed when plants experience low temperatures. They are characterized by a tighter hexacoordination than class 1 nsHbs and thus they have lower oxygen affinities (in the order of 100–200 nM). In Arabidopsis, AHb2 expression is resilient to hypoxic stress, but shows induction in response to low temperatures indicating a possible yet undefined role under cold-stress. AHb2 is also induced in response to the plant hormone cytokinin. The underlying mechanisms of AHb2-induced responses are still unresolved, and may differ from AHb1. Since the oxygen binding characteristics of AHb2 are comparable to leghemoglobin, a specific function of AHb2 in facilitating oxygen diffusion cannot be excluded. Although the existence of a mammalian type of nitric oxide synthase (NOS) remains controversial, NO appears to be a key signalling molecule in a broad array of pathophysiological and developmental events. The common metabolite nitrite is an alternative potential source of NO since it can be reduced to NO by a side-reaction of the cytosolic plant nitrate reductase, but also by plant mitochondria in the absence of oxygen. Nitrite and NO were recently shown to modulate oxygen balance and energy metabolism in developing seeds, suggesting the existence of a regulatory mechanism adjusting respiratory rate to prevent anoxia. The present project is divided as follows: (a) Effect of AHbs Pentacoordination on nitrite reductase activity. It has recently been reported that deoxy AHb1 and AHb2 reduce nitrite to form NO via a mechanism analogous to that observed for hemoglobin, myoglobin and neuroglobin. There is a possibility that plant Hbs produce NO in addition to scavenging it. Such a reaction could occur in plants because hypoxia leads to an increase in nitrite levels and NO emission, particularly in the dark. To test the hypothesis that a change in the equilibrium between six- and five-coordinate heme mediates the control in the nitrite reduction rate, we have generated distal pocket mutants of the two AHbs and the resulting proteins have been examined for nitrite reductase activity and spectroscopic features. Additionally, we have examined the effects of the active site residues on the ligand binding to the heme iron of AHb using UV-Vis spectroscopy in order to better understand the function of non-symbiotic hemoglobin from A.thaliana. We measured the absorption spectra over a wide range of pH which enabled us to examine the competition of endogenous and exogenous ligands for the sixth heme coordination as a function of the protonation states of the residues in the vicinity of the active site. Besides AHbs hexacoordination of the ferric and ferrous unligated species, it exhibits a few other remarkable features. In case of AHb2, a variety of different heme complexes exist in the ferric and ferrous states, and HisE7 (at position 66) and LysE10 (at position 69) can both compete as endogenous ligands. (b) The heme iron atom of AHb1 as substrate for a reductase partner. The reaction of nsHb with NO involves a change in heme-iron oxidation state. In NO dioxygenation (NOD) reaction oxyHb reacts with NO to yield nitrate and ferric Hb. Catalytic continuation of this reaction requires re-reduction of the heme iron. The most likely source of reduction is a reductase enzyme with specific activity toward the Hb. This idea has led to recent searches for Hb-specific reductases in plants, animals, and bacteria. However, such enzymes are difficult to identify because they likely dissociate after catalysis. Here, we have employed pull-down technique for protein-protein interaction to identify potential cognate reductase. Total protein was extracted from the leaves of A.thaliana (Col-0) and subjected to incubation with histidine tagged AHb1 bound with Ni2+-sepharose beads. The Prey protein (Cognate reductase) interaction with Bait (AHb1) was studied by loading the eluate on the 12% SDS-PAGE and subsequent identification of prey protein with mass-spectrometry. In a different approach, we used reduction system(s) (Monodehydroascorbate reductase and Glutathione reductase from A.thaliana) to test their efficiency to reduce the ferric heme iron in vitro. Monodehydroascorbate reductase (MDAR, EC 1.6.5.4) and Glutathione reductase (GR, EC 1.8.1.7) are key enzymes in ascorbate-glutathione cycle and are important to maintain the redox status of the plant cell. MDAR and GR contain FAD- and NADH-binding domains and have some similarity to the flavoprotein part of bacterial flavohemoglobins. We observed that MDAR (but not GR), along with ascorbate, effectively maintain the AHb1 WT in the reduced state.