The Mo(V) state of the molybdoenzyme sulfite oxidase (SO) is paramagnetic and can be studied by electron paramagnetic resonance (EPR) spectroscopy. Vertebrate SO at pH < 7 and pH > 9 exhibits characteristic EPR spectra that correspond to two structurally different forms of the Mo(V) active center referred to as the low-pH (lpH) and high-pH (hpH) forms, respectively. Both EPR forms have an exchangeable equatorial OH ligand, but its orientation in the two forms is different. It has been hypothesized that the formation of the lpH species is dependent upon the presence of chloride. In this work we have prepared and purified samples of wild type and various mutants of human SO that are depleted in chloride. These samples do not exhibit the typical lpH EPR spectrum at low pH, but rather show spectra that are characteristic of the blocked species that contains an exchangeable equatorial sulfate ligand. Addition of chloride to these samples results in the disappearance of the blocked species and the formation of the lpH species. Similarly, if chloride is added before sulfite, the lpH species is formed instead of the blocked one. Qualitatively similar results were observed for samples of sulfite oxidizing enzymes from other organisms that were previously reported to form a blocked species at low pH. However, the depletion of chloride has no effect upon the formation of the hpH species.The sulfite-oxidizing enzymes (SOEs), represented by sulfite oxidase (SO) in vertebrates and plants and sulfite dehydrogenase (SDH) in bacteria, catalyze the oxidation of sulfite to sulfate as represented by generic Eq. 1 (1).(1)In humans SO is essential for normal neonatal neurological development, and inborn deficiencies in SO result in severe physical and neurological disorders and early death (2,3).Reaction (1) is catalyzed by the square-pyramidal oxo-molybdenum active center, which has three equatorial sulfur ligands (one from the conserved cysteinyl side chain, and two from the molybdopterin cofactor), one axial oxo ligand, and an exchangeable equatorial oxo ligand in the solvent accessible pocket of the active site (4, 5). During the proposed catalytic cycle (6), sulfite initially reduces Mo(VI) to Mo(IV). Regeneration of the Mo(VI) state Unlike X-ray crystallography or extended X-ray absorption fine structure (EXAFS) spectroscopy, EPR can detect protons in the vicinity of a paramagnetic center and is able to unequivocally identify specific nuclei through using substitutions by or permutations of magnetic isotopes (e.g., 16 O → 17 O, 35 Cl → 37 Cl, 14 N → 15 N, etc.). Both, continuous wave (CW) and pulsed EPR spectroscopic approaches have been used to establish the effects of pH, anions in the media, and mutations near the active site on the identity and structure of the exchangeable equatorial ligand of the Mo(V) ion. It was found that in the absence of inhibiting anions (e.g., PO 4 3− , AsO 4 3− ), wild type (wt) vertebrate SO can show two distinct types of EPR signals, high-pH (hpH) and low-pH (lpH), corresponding to two ...

Green leaf volatiles (GLVs) are a diverse group of fatty acid-derived compounds emitted by all plants and are involved in a wide variety of developmental and stress-related biological functions. Recently, GLV emission bursts from leaves were reported following light-dark transitions and hypothesized to be related to the stress response while acetaldehyde bursts were hypothesized to be due to the 'pyruvate overflow' mechanism. In this study, branch emissions of GLVs and a group of oxygenated metabolites (acetaldehyde, ethanol, acetic acid, and acetone) derived from the pyruvate dehydrogenase (PDH) bypass pathway were quantified from mesquite plants following light-dark transitions using a coupled GC-MS, PTR-MS, and photosynthesis system. Within the first minute after darkening following a light period, large emission bursts of both C(5) and C(6) GLVs dominated by (Z)-3-hexen-1-yl acetate together with the PDH bypass metabolites are reported for the first time. We found that branches exposed to CO(2)-free air lacked significant GLV and PDH bypass bursts while O(2)-free atmospheres eliminated the GLV burst but stimulated the PDH bypass burst. A positive relationship was observed between photosynthetic activity prior to darkening and the magnitude of the GLV and PDH bursts. Photosynthesis under (13)CO(2) resulted in bursts with extensive labeling of acetaldehyde, ethanol, and the acetate but not the C(6)-alcohol moiety of (Z)-3-hexen-1-yl acetate. Our observations are consistent with (1) the "pyruvate overflow" mechanism with a fast turnover time (<1 h) as part of the PDH bypass pathway, which may contribute to the acetyl-CoA used for the acetate moiety of (Z)-3-hexen-1-yl acetate, and (2) a pool of fatty acids with a slow turnover time (>3 h) responsible for the C(6) alcohol moiety of (Z)-3-hexen-1-yl acetate via the 13-lipoxygenase pathway. We conclude that our non-invasive method may provide a new valuable in vivo tool for studies of acetyl-CoA and fatty acid metabolism in plants at a variety of spatial scales.

Prolonged drought stress combined with high leaf temperatures can induce programmed leaf senescence involving lipid peroxidation, and the loss of net carbon assimilation during early stages of tree mortality. Periodic droughts are known to induce widespread tree mortality in the Amazon rainforest, but little is known about the role of lipid peroxidation during drought-induced leaf senescence. In this study, we present observations of green leaf volatile (GLV) emissions during membrane peroxidation processes associated with the combined effects of high leaf temperatures and drought-induced leaf senescence from individual detached leaves and a rainforest ecosystem in the central Amazon. Temperature-dependent leaf emissions of volatile terpenoids were observed during the morning, and together with transpiration and net photosynthesis, showed a post-midday depression. This post-midday depression was associated with a stimulation of C5 and C6 GLV emissions, which continued to increase throughout the late afternoon in a temperature-independent fashion. During the 2010 drought in the Amazon Basin, which resulted in widespread tree mortality, green leaf volatile emissions (C6 GLVs) were observed to build up within the forest canopy atmosphere, likely associated with high leaf temperatures and enhanced drought-induced leaf senescence processes. The results suggest that observations of GLVs in the tropical boundary layer could be used as a chemical sensor of reduced ecosystem productivity associated with drought stress.

Although several per cent of net carbon assimilation can be re-released as isoprene emissions to the atmosphere by many tropical plants, much uncertainty remains regarding its biological significance. In a previous study, we detected emissions of isoprene and its oxidation products methyl vinyl ketone (MVK) and methacrolein (MACR) from tropical plants under high temperature/light stress, suggesting that isoprene is oxidized not only in the atmosphere but also within plants. However, a comprehensive analysis of the suite of isoprene oxidation products in plants has not been performed and production relationships with environmental stress have not been described. In this study, putative isoprene oxidation products from mango (Mangifera indica) branches under abiotic stress were first identified. High temperature/light and freeze–thaw treatments verified direct emissions of the isoprene oxidation products MVK and MACR together with the first observations of 3-methyl furan (3-MF) and 2-methyl-3-buten-2-ol (MBO) as putative novel isoprene oxidation products. Mechanical wounding also stimulated emissions of MVK and MACR. Photosynthesis under 13CO2 resulted in rapid (<30min) labelling of up to five carbon atoms of isoprene, with a similar labelling pattern observed in the putative oxidation products. These observations highlight the need to investigate further the mechanisms of isoprene oxidation within plants under stress and its biological and atmospheric significance.