Electrochemical Kinetics Support a Second Coordination Sphere Mechanism in Metal‐Based Formate Dehydrogenase

International audienceMetal-based formate dehydrogenases are molybdenum or tungsten-dependent enzymes that catalyze the interconversion between formate and CO 2. According to the current consensus, the metal ion of the catalytic center in its active form is coordinated by 6 S (or 5 S and 1 Se) atoms, leaving no free coordination sites to which formate could bind to the metal. Some authors have proposed that one of the active site ligands decoordinates during turnover to allow formate binding. Another proposal is that the oxidation of formate takes place in the second coordination sphere of the metal. Here, we have used electrochemical steady-state kinetics to elucidate the order of the steps in the catalytic cycle of two formate dehydrogenases. Our results strongly support the "second coordination sphere" hypothesis

Identification and characterization of a non-canonical menaquinone-linked formate dehydrogenase

International audienceThe Molybdenum/Tungsten-bispyranopterin guanine dinucleotides (Mo/W-bisPGD) family of Formate Dehydrogenases (FDHs) plays roles in several metabolic pathways ranging from carbon fixation to energy harvesting owing to their reaction with a wide variety of redox partners. Indeed, this metabolic plasticity results from the diverse structures, cofactor content, and substrates employed by partner subunits interacting with the catalytic hub. Here, we unveiled two non-canonical FDHs in Bacillus subtilis which are organized into two-subunit complexes with unique features, ForCE1 and ForCE2. We show that the ForC catalytic subunit interacts with an unprecedented partner subunit, ForE, and that its amino acid sequence within the active site deviates from the consensus residues typically associated with FDH activity, as a histidine residue is naturally substituted with a glutamine. The ForE essential subunit mediates the utilization of menaquinone as an electron acceptor as shown by the formate:menadione oxidoreductase activity of both enzymes, their copurification with menaquinone, and the distinctive detection of a protein-bound neutral menasemiquinone radical by multifrequency electron paramagnetic resonance (EPR) experiments on the purified enzymes. Moreover, EPR characterization of both FDHs reveals the presence of several [Fe-S] clusters with distinct relaxation properties and a weakly anisotropic Mo(V) EPR signature, consistent with the characteristic Mo/bisPGD cofactor of this enzyme family. Altogether, this work enlarges our knowledge of the FDH family by identifying a non-canonical FDH, which differs in terms of architecture, amino acid conservation around the Mo cofactor, and reactivity

A new reservoir-based CPAP with low oxygen consumption: the Bag-CPAP

Abstract Background Several noninvasive ventilatory supports rely in their design on high oxygen consumption which may precipitate oxygen shortage, as experienced during the COVID-19 pandemic. In this bench-to-bedside study, we assessed the performance of a new continuous positive airway pressure (CPAP) device integrating a large reservoir (“Bag-CPAP”) designed to minimize oxygen consumption, and compared it with other CPAP devices. Methods First, a bench study compared the performances of Bag-CPAP and four CPAP devices with an intensive care unit ventilator. Two FiO2 targets (40–60% and 80–100%) at a predefined positive end expiratory pressure (PEEP) level between 5 and 10 cm H2O were tested and fraction of inspired oxygen (FiO2) and oxygen consumption were measured. Device-imposed work of breathing (WOB) was also evaluated. Second, an observational clinical study evaluated the new CPAP in 20 adult patients with acute respiratory failure in two hospitals in France. Actual FiO2, PEEP, peripheral oxygen saturation, respiratory rate, and dyspnea score were assessed. Results All six systems tested in the bench study reached the minimal FiO2 target of 40% and four reached at least 80% FiO2 while maintaining PEEP in the predefined range. Device-delivered FiO2/consumed oxygen ratio was the highest with the new reservoir-based CPAP irrespective of FiO2 target. WOB induced by the device was higher with Bag-CPAP. In the clinical study, Bag-CPAP was well tolerated and could reach high (> 90%) and moderate (> 50%) FiO2 with an oxygen flow rate of 15 [15–16] and 8 [7–9] L/min, respectively. Dyspnea score improved significantly after introduction of Bag-CPAP, and SpO2 increased. Conclusions In vitro, Bag-CPAP exhibited the highest oxygen saving properties albeit had increased WOB. It was well accepted clinically and reduced dyspnea. Bag-CPAP may be useful to treat patients with acute respiratory failure in the field, especially when facing constraints in oxygen delivery