The Heme Environment in Barley Hemoglobin

Abstract

To elucidate the environment and ligand structure of the heme in barley hemoglobin (Hb), resonance Raman and electron paramagnetic resonance spectroscopic studies have been carried out. The heme is shown to have bis-imidazole coordination, and neither of the histidines has imidazolate character. Barley Hb has a unique heme environment as judged from the Fe-CO and C-O stretching frequencies in the CO complex. Two Fe-CO stretching modes are observed with frequencies at 534 and 493 cm ؊1 , with relative intensities that are pH sensitive. The 534 cm ؊1 conformer shows a deuterium shift, indicating that the iron-bound CO is hydrogenbonded, presumably to the distal histidine. A C-O stretching mode at 1924 cm ؊1 is assigned as being associated with the 534 cm ؊1 conformer. Evidence is presented that the high Fe-CO and low C-O stretching frequencies (534 and 1924 cm ؊1 , respectively) arise from a short hydrogen bond between the distal histidine and the CO. The 493 cm ؊1 conformer arises from an open conformation of the heme pocket and becomes the dominant population under acidic conditions when the distal histidine moves away from the CO. Strong hydrogen bonding between the bound ligand and the distal histidine in the CO complex of barley Hb implies that a similar structure may occur in the oxy derivative, imparting a high stability to the bound oxygen. This stabilization is confirmed by the dramatic decrease in the oxygen dissociation rate compared with sperm whale myoglobin.Barley (Hordeum sp.) hemoglobin (Hb), 1 the first nonsymbiotic plant Hb to be isolated and characterized from a monocotyledonous plant (1), is expressed in seed and root tissues under anaerobic conditions. Because the level of Hb in barley aleurone tissue is of the order of only 20 M, an expression system in Escherichia coli was developed to produce a barley Hb fusion protein that is indistinguishable from the native protein in most properties but differs in having five extra amino acids at the N terminus (1).Nonsymbiotic plant Hbs constitute a new class of protoheme proteins that are expressed at low concentrations (on the order of 1-20 mol/kg wet tissue weight) in roots, stems, or germinating seeds of monocotyledonous (2, 3) and dicotyledenous plants (3-6). Nonsymbiotic Hbs of both monocots and dicots fall into a single coherent gene family, distinct from the family of genes that encode the symbiotic Hbs (6). They are functionally distinct from the familiar symbiotic plant leghemoglobins that are expressed by some plants in nitrogen-fixing symbiotic associations with the bacterium Rhizobium or the actinomycete Frankia. Distinguishing characteristics of nonsymbiotic Hbs (1, 7, 8) are extremely slow dissociation of bound oxygen and 6-coordinate, low spin ferrous (deoxy) species.The barley nonsymbiotic Hb gene is induced under conditions of low oxygen pressure (2). Low oxygen tension, per se, is not responsible for the induction, because the gene can be induced in the presence of oxygen by respiratory inhibitors that interfere with mitochondrial AT...

The Heme Environment of Mouse Neuroglobin

“…1). The 3 line, which is coordination-and spin state-sensitive, is detected in the 1470 -1505 cm Ϫ1 range for various ferric heme proteins (5,6,8). For Ngb, the frequency of the 3 line at 1505 cm Ϫ1 indicates that the heme is six-coordinate and low spin.…”

“…Globins of vertebrates, like Mb and Hb, are normally six-coordinate and high spin in the ferric state, with a water molecule coordinated to the iron and five-coordinate in the ferrous form, which leaves the sixth position empty and ready for the binding of exogenous ligands such as CO and oxygen. However, hexacoordinate low spin forms of ferric and ferrous globins are frequently observed for the globins of invertebrates (12,13) and plants (8,14). As might be expected, for several of these hexacoordinate heme proteins, the presence of a sixth ligand to the iron interferes with exogenous ligand binding (8,13,15 2 for both human and mouse Ngb.…”

“…However, hexacoordinate low spin forms of ferric and ferrous globins are frequently observed for the globins of invertebrates (12,13) and plants (8,14). As might be expected, for several of these hexacoordinate heme proteins, the presence of a sixth ligand to the iron interferes with exogenous ligand binding (8,13,15 2 for both human and mouse Ngb. These on-rates for Ngb are consistent with the conclusions drawn from our Raman results based on the absence of strain in the Fe-His bond.…”

“…The frequency of the 4 line for Ngb is typical of ferrous forms of heme proteins (7). The frequency of the 3 line at 1493 cm Ϫ1 indicates that the heme is six-coordinate and low spin (5,8,9). The low frequency region of the resonance Raman spectra of heme proteins contains heme-ligand vibrational modes that allow the identification of the axial ligands of the heme (5, 7).…”

Modeling the tertiary structure of a maize (Zea mays ssp. mays) non-symbiotic hemoglobin

“…The best characterized nsHbs are rice Hb1 and barley Hb [5,8,11,14,15]; however, limited structural analysis has been performed on other plant nsHbs. In this work we have utilized computational methods to predict the tertiary structure of a Hb from maize (Hbm) [3].…”