The structures and vibrational frequencies of the acetate ion interacting with a metal ion (Na + , Mg 2+ , and Ca 2+ ) in the unidentate, bidentate, bridging, and pseudobridging forms are studied by ab initio molecular orbital calculations. Effects of a water molecule coordinating to either the acetate ion or the metal ion are also examined. The calculations are carried out by using the self-consistent reaction field method at the Hartree-Fock level with the 6-31+G** basis set. For the species interacting with a divalent metal cation, the lengths of the two CO bonds of the acetate ion are nearly equal in the bidentate form but are significantly different in the unidentate form. The frequency of the COO -antisymmetric stretch of the unidentate species is higher than that of the ionic species, which is in turn higher than that of the bidentate species. The reverse is the case for the COO -symmetric stretch. As a result, the frequency separations (∆ν a-s ) between the COO -antisymmetric and symmetric stretches for the unidentate, bidentate, and ionic species are in the following order: ∆ν a-s (unidentate) > ∆ν a-s (ionic) > ∆ν a-s (bidentate). It is demonstrated that such a correlation between the vibrational frequencies of the COO -group and the types of its coordination to a divalent metal cation is related to changes in the CO bond lengths and the OCO angle. The results of the present study clarify the physical basis of the empirical structure-frequency correlation, which has been used in the analysis of the infrared spectra of Ca 2+ -binding proteins.
Plant respiratory burst oxidase homolog (rboh) proteins, which are homologous to the mammalian 91-kDa glycoprotein subunit of the phagocyte oxidase (gp91 phox ) or NADPH oxidase 2 (NOX2), have been implicated in the production of reactive oxygen species (ROS) both in stress responses and during development. Unlike mammalian gp91 phox /NOX2 protein, plant rboh proteins have hydrophilic N-terminal regions containing two EF-hand motifs, suggesting that their activation is dependent on Ca 2؉ . However, the significance of Ca 2؉ binding to the EF-hand motifs on ROS production has been unclear. By employing a heterologous expression system, we showed that ROS production by Arabidopsis thaliana rbohD (AtrbohD) was induced by ionomycin, which is a Ca 2؉ ionophore that induces Ca 2؉ influx into the cell. This activation required a conformational change in the EF-hand region, as a result of Ca 2؉ binding to the EF-hand motifs. We also showed that AtrbohD was directly phosphorylated in vivo, and that this was enhanced by the protein phosphatase inhibitor calyculin A (CA). Moreover, CA itself induced ROS production and dramatically enhanced the ionomycin-induced ROS production of AtrbohD. Our results suggest that Ca 2؉ binding and phosphorylation synergistically activate the ROS-producing enzyme activity of AtrbohD.Photosynthetic plants have developed various mechanisms to cope with oxidative stress, such as the production of antioxidants and enzymes that scavenge reactive oxygen species (ROS).3 Plants are also equipped with mechanisms for producing ROS in response to internal and external stimuli. ROS production is induced during many physiological processes, including stress responses, cell growth, hormonal responses, stomatal closure, and disease resistance (see Refs. 1-4 and references therein).ROS production is induced in plants in response to recognition of pathogenic signals, such as pathogen/microbe-associated molecular patterns (PAMPs/MAMPs) or elicitors. Elicitor-induced ROS production is preceded by a rapid increase in the cytosolic free Ca 2ϩ concentration ([Ca 2ϩ ] cyt ) (5-7) and is inhibited both by Ca 2ϩ chelators such as EGTA and BAPTA, and by Ca 2ϩ channel blockers such as La 3ϩ (6,8). The overexpression of rice two-pore channel 1 (OsTPC1), which is a putative voltage-gated Ca 2ϩ channel, enhanced elicitor-induced ROS production (9). Elicitor-induced ROS production is also inhibited by diphenylene iodonium (DPI), which is known to inhibit NADPH oxidase activity (6, 10). NADPH oxidase activity in the microsomal membrane fraction from tomato and tobacco was activated by adding Ca 2ϩ in vitro (11), suggesting that elicitor-induced ROS production by plant NADPH oxidase might be dependent on Ca 2ϩ . In mammalian phagocytes, ROS production is mediated by the NADPH-dependent phagocytic oxidase (phox) complex, which consists of the catalytic subunit gp91 phox /NADPH oxidase (NOX) 2, together with the regulatory subunits p22 phox , p40 phox , p47 phox , p67 phox , and the small GTP-binding protein Rac (12). In...
We review the Fourier-transform infrared (FTIR) spectroscopy of side-chain COO(-) groups of Ca(2+)-binding proteins: parvalbumins, bovine calmodulin, akazara scallop troponin C and related calcium binding proteins and peptide analogues. The COO(-) stretching vibration modes can be used to identify the coordination modes of COO(-) groups of Ca(2+)-binding proteins to metal ions: bidentate, unidentate, and pseudo-bridging. FTIR spectroscopy demonstrates that the coordination structure of Mg(2+) is distinctly different from that of Ca(2+) in the Ca(2+)-binding site in solution. The interpretation of COO(-) stretches is ensured on the basis of the spectra of calcium-binding peptide analogues. The implication of COO(-) stretches is discussed for Ca(2+)-binding proteins. This article is part of a Special Issue entitled: FTIR in membrane proteins and peptide studies.
Metal-ligand interactions in the Ca"-binding sites of pike parvalbumin (PI = 4.10) have been examined by Fourier-transform infrared spectroscopy. The region of the COO-antisymmetric stretch provides useful information on the types of coordination of the COO-groups to the metal ions in the Mg"-, Mn"-, and Ca"-bound forms. In the spectrum of the Ca*'-bound form, two bands are observed at 1,582 and 1,553 cm-', whereas, in the spectra of the Me-and Mn"-bound forms, bands are observed only in the region around 1,582 cm-' and no band is found in the region around 1,553 cm-'. The 1,553-cm-' band of the Ca*'-bound form reflects the bidentate coordination of the COO-groups of both Glu-62 in the CD site and Glu-101 in the EF site to the Ca" ions, which has been made clear by X-ray analysis as a feature of the Ca"-bound form. Absence of such a band in the spectrum of the Mn"-bound form is consistent with the X-ray structure of this form where both of the two COO-groups are unidentate. These unidentate COO-groups of Glu-62 and Glu-101 in the Mn"-bound form seem to give rise to a band at 1,577-1,574 cm-'. The spectrum of the Mg"-bound form is also consistent with the 'pseudo-bridging' coordination of the COO-group of Glu-101 reported in the X-ray structure of a form where the Mg2+ ion occupies only the EF site, and the same spectrum is further indicative of the 'pseudo-bridging' coordination of the COOgroup of Glu-62.
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