Formate oxidation to carbon dioxide is a key reaction in one-carbon compound metabolism, and its reverse reaction represents the first step in carbon assimilation in the acetogenic and methanogenic branches of many anaerobic organisms. The molybdenum-containing dehydrogenase FdsABG is a soluble NAD+-dependent formate dehydrogenase and a member of the NADH dehydrogenase superfamily. Here, we present the first structure of the FdsBG subcomplex of the cytosolic FdsABG formate dehydrogenase from the hydrogen-oxidizing bacterium Cupriavidus necator H16 both with and without bound NADH. The structures revealed that the two iron-sulfur clusters, Fe4S4 in FdsB and Fe2S2 in FdsG, are closer to the FMN than they are in other NADH dehydrogenases. Rapid kinetic studies and EPR measurements of rapid freeze-quenched samples of the NADH reduction of FdsBG identified a neutral flavin semiquinone, FMNH•, not previously observed to participate in NADH-mediated reduction of the FdsABG holoenzyme. We found that this semiquinone forms through the transfer of one electron from the fully reduced FMNH−, initially formed via NADH-mediated reduction, to the Fe2S2 cluster. This Fe2S2 cluster is not part of the on-path chain of iron-sulfur clusters connecting the FMN of FdsB with the active-site molybdenum center of FdsA. According to the NADH-bound structure, the nicotinamide ring stacks onto the re-face of the FMN. However, NADH binding significantly reduced the electron density for the isoalloxazine ring of FMN and induced a conformational change in residues of the FMN-binding pocket that display peptide-bond flipping upon NAD+ binding in proper NADH dehydrogenases.
We aim to use the resolving power of near-infrared (NIR) fluorescence lifetime microscopy (FLIM) to provide information on the fluorescence decay behavior of NIR FRET donor probes, both in solution and in vitro, and assess their impact on in vivo macroscopic FLI FRET (MFLI FRET) tumor imaging. Utilizing HER2 mAbs, i.e., trastuzumab (TZM), labeled with AlexaFluor 700 (AF700), and HER2 positive cancer cell lines (AU565 and SKOV-3), we have documented significant impacts of IRF extraction methods and probe labeling schemes on FLIM analysis. Additionally, we have noted marked variation in the intracellular distribution of the HER2-TZM binding complexes, as well as in average endosomal lifetime measurements between cell lines. Herein, we discuss optimal methods for IRF extraction and generating NIR probes, as well as results from the newly optimized NIR FLIM FRET assay demonstrating variations in the average intracellular lifetime of TZM-AF700. Because fluorescence lifetime is impacted by environmental factors, such as pH, refraction, viscosity, and proximity to other molecules, these variations imply differences in the way TZM interacts with the endosomal microenvironment of these cell lines. We hypothesize that different HER2 positive cancer types exhibit variations in endosomal trafficking of the HER2-drug complex that play a key role in primary/acquired resistance to TZM.
Soluble formate dehydrogenase FdsABG, is a member of the NADH dehydrogenase superfamily, which is one of the most broadly distributed family of enzymes. Unlike most members of this superfamily, formate dehydrogenase FdsABG is a cytosolic enzyme. Physiologically, the FdsABG from Cupriavidus necator oxidizes formate to CO2 by reducing NAD+ to NADH. In the process, reducing equivalents are transferred from formate to the molybdenum center of FdsA, from where they are passed through a chain of iron‐sulfur clusters to the FMN of FdsB and on to NAD+. These two steps of accepting reducing equivalents from the formate and passing them on to NAD+ are considered the reductive and oxidative half‐reactions of the formate dehydrogenase reaction. Because FdsB‐ and FdsG‐like domains are found in all NADH dehydrogenases and can perform the oxidative half reaction independent of FdsA, we embarked on a crystallographic study of FdsBG of C. necator formate dehydrogenase, with the aim of putting the oxidative half reaction of the enzyme in a structural context. Support or Funding Information United States Department of Energy Grant DE‐SC0010666 (to R. H.)
The soluble molybdenum‐containing, NAD+‐dependent formate dehydrogenase FdsDABG from Cupriavidus necator belongs to the NADH dehydrogenase superfamily and catalyzes the oxidation of formate to CO2 and the reduction of NAD+ to NADH. Here, we present the first description of the crystal structure of the FdsBG subcomplex with and without bound NADH. Compared to other NADH dehydrogenases, FMN is closer to both iron‐sulfur clusters, Fe4S4 in FdsB and Fe2S2 in FdsG. Based on the NADH‐bound structure, we conclude that the nicotinamide ring of NADH can only access the re‐face of FMN. However, the binding of NADH reduces the affinity of the isoalloxazine ring of FMN and allows for a conformational change of the residues that are known to undergo an oxidation state‐dependent peptide flip in canonical NADH dehydrogenases.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.