Cytochrome bd–type quinol oxidases catalyze the reduction of molecular oxygen to water in the respiratory chain of many human-pathogenic bacteria. They are structurally unrelated to mitochondrial cytochrome c oxidases and are therefore a prime target for the development of antimicrobial drugs. We determined the structure of the Escherichia coli cytochrome bd-I oxidase by single-particle cryo–electron microscopy to a resolution of 2.7 angstroms. Our structure contains a previously unknown accessory subunit CydH, the L-subfamily–specific Q-loop domain, a structural ubiquinone-8 cofactor, an active-site density interpreted as dioxygen, distinct water-filled proton channels, and an oxygen-conducting pathway. Comparison with another cytochrome bd oxidase reveals structural divergence in the family, including rearrangement of high-spin hemes and conformational adaption of a transmembrane helix to generate a distinct oxygen-binding site.

Pathophysiologies of cancer-associated syndromes such as cachexia are poorly understood and no routine biomarkers have been established, yet. Using shotgun proteomics, known marker molecules including PMEL, CRP, SAA, and CSPG4 were found deregulated in patients with metastatic melanoma. Targeted analysis of 58 selected proteins with multiple reaction monitoring was applied for independent data verification. In three patients, two of which suffered from cachexia, a tissue damage signature was determined, consisting of nine proteins, PLTP, CD14, TIMP1, S10A8, S10A9, GP1BA, PTPRJ, CD44, and C4A, as well as increased levels of glycine and asparagine, and decreased levels of polyunsaturated phosphatidylcholine concentrations, as determined by targeted metabolomics. Remarkably, these molecules are known to be involved in key processes of cancer cachexia. Based on these results, we propose a model how metastatic melanoma may lead to reprogramming of organ functions via formation of platelet activating factors from long-chain polyunsaturated phosphatidylcholines under oxidative conditions and via systemic induction of intracellular calcium mobilization. Calcium mobilization in platelets was demonstrated to alter levels of several of these marker molecules. Additionally, platelets from melanoma patients proved to be in a rather exhausted state, and platelet-derived eicosanoids implicated in tumor growth were found massively increased in blood from three melanoma patients. Platelets were thus identified Serum is the most important diagnostic sample type because of its minimal invasive access, a relatively high stability and its comprehensive representation of the physiological state of an individual. The latest technological development of mass spectrometric instruments, such as high resolution orbitrap instruments, improved substantially the quality and reliability of serum proteomics data (1). Shotgun proteomics analysis using the orbitrap technology is mainly applied for protein screening purposes (2). This method allows performing untargeted analyses, applicable for hypothesis-generating clinical applications. Targeted proteomics using triple-quadrupole mass spectrometric instruments, represents a complementary approach which is applied for protein quantification, hypotheses verification and validation (3). Enormous efforts have been invested in the last decades focusing on serum biomarker discovery (4). However, mass spectrometrybased proteomics analyses hardly managed to cope with biological variation. As a consequence, almost no clinically validated biomarker has emerged from these investigations yet, and a lot of questions about pathophysiological mechanisms remain unanswered.Using mass spectrometry-based proteomics, we have previously investigated melanoma and neighboring stroma cells, focusing on the identification of biomarkers associated with intrinsic and extrinsic drug resistance (5). In the present article, we followed the question how metastatic melanoma may reprogram distant organ functions, and how these...

Na/H antiporters comprise a family of membrane proteins evolutionarily conserved in all kingdoms of life and play an essential role in cellular ion homeostasis. The NhaA crystal structure of has become the paradigm for this class of secondary active transporters. However, structural data are only available at low pH, where NhaA is inactive. Here, we adapted hydrogen/deuterium-exchange mass spectrometry (HDX-MS) to analyze conformational changes in NhaA upon Li binding at physiological pH. Our analysis revealed a global conformational change in NhaA with two sets of movements around an immobile binding site. Based on these results, we propose a model for the ion translocation mechanism that explains previously controversial data for this antiporter. Furthermore, these findings contribute to our understanding of related human transporters that have been linked to various diseases.

Flavin-based electron bifurcation (FBEB) is a novel mechanism of energy coupling used by anaerobic microorganisms to optimize their energy metabolism efficiency. The first high-resolution structure of a complete FBEB enzyme complex, the NADH-dependent reduced ferredoxin: NADP -oxidoreductase (NfnAB) of Thermotoga maritima, was recently solved. However, no experimental evidence for the NADPH-binding site and conformational changes during the FBEB reaction are available. Here we analyzed ligand binding and the conformational dynamics of oxygen-sensitive NfnAB using Hydrogen-Deuterium Exchange Mass-Spectrometry, including a customized anaerobic workflow. We confirmed the NADH and the previously postulated NADPH-binding site. Furthermore, we observed an NfnA-NfnB rearrangement upon NADPH binding which supports the proposed FBEB mechanism.

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