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Superoxide is a reactive oxygen species produced during aerobic metabolism in mitochondria and prokaryotes. It causes damage to lipids, proteins and DNA and is implicated in cancer, cardiovascular disease, neurodegenerative disorders and aging. As protection, cells express soluble superoxide dismutases, disproportionating superoxide to oxygen and hydrogen peroxide. Here, we describe a membrane-bound enzyme that directly oxidizes superoxide and funnels the sequestered electrons to ubiquinone in a diffusion-limited reaction. Experiments in proteoliposomes and inverted membranes show that the protein is capable of efficiently quenching superoxide generated at the membrane in vitro. The 2.0 Å crystal structure shows an integral membrane di-heme cytochrome b poised for electron transfer from the P-side and proton uptake from the N-side. This suggests that the reaction is electrogenic and contributes to the membrane potential while also conserving energy by reducing the quinone pool. Based on this enzymatic activity, we propose that the enzyme family be denoted superoxide oxidase (SOO).

Cytochrome c oxidase is a multisubunit membrane-bound enzyme, which catalyzes oxidation of four molecules of cytochrome c2+ and reduction of molecular oxygen to water. The electrons are taken from one side of the membrane while the protons are taken from the other side. This topographical arrangement results in a charge separation that is equivalent to moving one positive charge across the membrane for each electron transferred to O2. In this reaction part of the free energy available from O2 reduction is conserved in the form of an electrochemical proton gradient. In addition, part of the free energy is used to pump on average one proton across the membrane per electron transferred to O2. Our understanding of the molecular design of the machinery that couples O2 reduction to proton pumping in oxidases has greatly benefited from studies of so called "uncoupled" structural variants of the oxidases. In these uncoupled oxidases the catalytic O2-reduction reaction may display the same rates as in the wild-type CytcO, yet the electron/proton transfer to O2 is not linked to proton pumping. One striking feature of all uncoupled variants studied to date is that the (apparent) pKa of a Glu residue, located deeply within a proton pathway, is either increased or decreased (from 9.4 in the wild-type oxidase). The altered pKa presumably reflects changes in the local structural environment of the residue and because the Glu residue is found near the catalytic site as well as near a putative exit pathway for pumped protons these changes are presumably important for controlling the rates and trajectories of the proton transfer. In this paper we summarize data obtained from studies of uncoupled structural oxidase variants and present a hypothesis that in quantitative terms offers a link between structural changes, modulation of the apparent pKa and uncoupling of proton pumping from O2 reduction.

Large amounts of methane (CH4) are known to be emitted from permafrost environments during the autumn freeze‐in, but the specific soil conditions leading up to these bursts are unclear. Therefore, we used an ultrawide band ground‐penetrating radar in Northeast Greenland in autumn 2009 to estimate the volumetric composition inside the soil through dielectric characterization from 200 to 3200 MHz. Our results suggest a compression of the gas reservoir during the phase transition of soil water, which is accompanied by a peak in surface CH4 emissions. About 1 week thereafter, there seems to be a decompression event, consistent with ground cracking which allows the gas reservoir to expand again. This coincides with the largest CH4 emission, exceeding the summer maximum by a factor of 4. We argue that these complementary measurement techniques are needed to come to an understanding of tundra CH4 bursts connected to soil freezing.

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BackgroundPain is a common condition among prehospital patients. The present study is designed to determine whether adding low-dose ketamine as additional analgesia improves the pain/nausea scores and hemodynamic parameters compared to morphine sulphate alone among patients with bone fractures.MethodsProspective, prehospital clinical cohort study. Twenty-seven patients were included with acute pain. Eleven patients received morphine sulphate 0.2 mg/kg (M-group) and 16 patients received morphine sulphate 0.1 mg/kg combined with 0.2 mg/kg ketamine (MK-group). Scores for pain, nausea, sedation (AVPU) and the haemodynamic parameters (systolic blood pressures (BP), heart rate (HR) and peripheral oxygen saturation (SpO2) were recorded at rescue scene before the start of analgesia and subsequently to admission at hospital.ResultsMean treatment time 46 ± 17 minutes in the M-group and 56 ± 11 minutes in the MK-group, respectively (ns). Mean doses of morphine sulphate in the M-group were 13.5 ± 3.2 mg versus 7.0 ± 1.5 mg in the MK-group. The mean additional doses of ketamine in the MK-group were 27.9 ± 11.4 mg. There were significantly differences between the M- and the MK-group according to NRS scores for pain (5.4 ± 1.9 versus 3.1 ± 1.4) and BP (134 ± 21 mmHg versus 167 ± 32 mmHg) at admission at hospital, respectively (P < 0.05). All patients were Alert or respond to Voice and the results were similar between the groups. One patient versus 4 patients reported nausea in the M- and MK-group, respectively, and 3 patients vomited in the Mk-group (ns).ConclusionWe conclude that morphine sulphate with addition of small doses of ketamine provide adequate pain relief in patients with bone fractures, with an increase in systolic blood pressure, but without significant side effects.

Cytochrome c oxidase (CytcO) is a membrane-bound enzyme, which catalyzes the reduction of di-oxygen to water and uses a major part of the free energy released in this reaction to pump protons across the membrane. In the Rhodobacter sphaeroides aa3 CytcO all protons that are pumped across the membrane, as well as one half of the protons that are used for O2 reduction, are transferred through one specific intraprotein proton pathway, which holds a highly conserved Glu286 residue. Key questions that need to be addressed in order to understand the function of CytcO at a molecular level are related to the timing of proton transfers from Glu286 to a “pump site” and the catalytic site, respectively. Here, we have investigated the temperature dependencies of the H/D kinetic-isotope effects of intramolecular proton-transfer reactions in the wild-type CytcO as well as in two structural CytcO variants, one in which proton uptake from solution is delayed and one in which proton pumping is uncoupled from O2 reduction. These processes were studied for two specific reaction steps linked to transmembrane proton pumping, one that involves only proton transfer (peroxy–ferryl, P→F, transition) and one in which the same sequence of proton transfers is also linked to electron transfer to the catalytic site (ferryl–oxidized, F→O, transition). An analysis of these reactions in the framework of theory indicates that that the simpler, P→F reaction is rate-limited by proton transfer from Glu286 to the catalytic site. When the same proton-transfer events are also linked to electron transfer to the catalytic site (F→O), the proton-transfer reactions are gated by a protein structural change, which presumably ensures that the proton-pumping stoichiometry is maintained also in the presence of a transmembrane electrochemical gradient.