Insights Into the Proton Translocation Pathway of the Organohalide Respiratory Complex of Dehalococcoides mccartyi Strain CBDB1
Nadine Hellmold1, Marie Eberwein1, Darja Deobald1, Lorenz Adrian1,2
1 Helmholtz Centre for Environmental Research – UFZ, Permoserstraße 15, Leipzig, Germany
2 Institute of Biotechnology, Chair of Geobiotechnology, Technische Universität Berlin, Berlin, Germany
Introduction: Dehalococcoides mccartyi CBDB1, a strictly anaerobic bacterium of the Chloroflexi phylum uses hydrogen as sole electron donor and different organohalides as terminal electron acceptors in a process called organohalide respiration (OHR). Dehalogenation is catalyzed by a corrinoid-dependent reductive dehalogenase (RdhA) that is part of a membrane-bound OHR protein complex, composed of at least seven proteins: RdhA and its anchor protein RdhB, hydrogenase subunits HupL and HupS, ferredoxin-like protein HupX, OmeA, and the membrane-bound subunit OmeB. Notably, the OHR complex lacks quinones, cytochromes and proton pumps. Here, we developed an in-vitro enzyme activity assay using deuterium-labeled water (D2O) and methyl viologen as artificial electron donor allowing to spatially track the incorporation of protons into the dehalogenation product.
Methods: In-vitro dehalogenase activity assays were conducted anaerobically with CBDB1 cells cultivated in H2O- or D2O-containing medium, and reaction mixes with D2O or H2O, to have either D2O outside and H2O inside the cells, or vice versa. We optimized the activity assay and termination procedure to ensure accurate quantification of dehalogenation products by GC-MS before diffusion equilibrium across the membrane was reached. Then, the ‘deuterium degree’ was determined describing the relative proportion of deuterated products. To predict the proton path across the membrane, we performed a multiple sequence alignment of OmeB and homologous NrfD-like proteins from various bacteria using the MUSCLE algorithm in MEGA11 and calculated the structure of the OmeA/OmeB/HupX submodule and of RdhA using AlphaFold2 ColabFold.
Preliminary results: During the reductive dehalogenation reaction, the substrate is protonated. To analyze if the incorporated proton originates from the inside (cytoplasm) or outside (exoplasm) of the cell, we determined the deuterium degree in the resulting product. Our experimental data with D2O initially outside and H2O inside the cells, show an increase of the deuterium degree towards an equilibrium over time, indicating the initial incorporation of protons. The converse experiment with D2O inside and H2O outside the cells shows a decreasing deuterium degree over time. Although this second data set is less pronounced, it indicates initial incorporation of deuterium ions. Both experiments therefore suggest that protons are passed through the membrane directly onto the substrate. To identify a potential proton pathway through the protein complex, we analyzed the core structures of the complex. Structural and sequential analysis revealed that OmeB, a member of the NrfD-like protein group, contains several conserved, mostly charged amino acids. The protein structure of OmeB also features a prominent quinone binding site (Q-site), covered by the lower part of HupX, while OmeA represents the uppermost part of the subcomplex. The Fe-S clusters and molybdenum cofactors in HupX and OmeA are arranged linearly within the subcomplex, converging at the putative Q-site in OmeB. We manually docked RdhA to the OmeA/OmeB/HupX subcomplex, with its Fe-S cluster less than 10 Å distant from the membrane-nearest Fe-S cluster of HupX, in close proximity to the putative Q-site of OmeB. In the absence of quinones, RdhA and its substrate appear to take over the quinone’s function. Thus, the dehalogenation reaction contributes directly to the proton motive force by transferring protons from the cytoplasm to the substrate. Charged amino acids distributed in the membrane helices of OmeB may be involved in proton translocation, as suggested for other NrfD-like proteins
Novel aspect: Combining experimental and structural data we propose how quinone-independent protein-based organohalide respiration could couple electron flux with proton translocation.
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