Dehalococcoides mccartyi is a slow-growing, strictly anaerobic bacterium that uses persistent halogenated hydrocarbons as terminal electron acceptors in its respiration. This respiration is catalysed by a membranebound, multi-subunit organohalide respiration (OHR) complex (~340 kDa). The key enzyme in the OHR is the reductive dehalogenase (RDase), which catalyses the final electron transfer to the substrate. No quinones, cytochromes or proton channels are involved in this process. Therefore, it has been unclear how Dehalococcoides strains generate a proton motive force (pmf) across the membrane. The genetic inaccessibility of the organism and a so far lack of heterologous expression of the entire complex have hindered the structural and biochemical characterisation of the OHR complex. In this work, the reductive dehalogenation activity of D. maccartyi strain CBBD1 was investigated under specific conditions to test whether the protons for the halogen substitution during respiration originate from inside or outside the cell. In addition, a computational model of the entire stand-alone OHR complex was developed. Different purification strategies were tested to enable structural characterisation of the OHR complex derived from wild-type cells on the long run. By using a deuterium-based activity assay, evidence was obtained that protons from the cell interior are transferred to the electron acceptor substituting the halogen atom. This mechanism couples the external electron flow to the necessary proton flux from the cytoplasm to the periplasm, generating the pmf. Using computational and evolutionary analyses, the largest transmembrane subunit of the complex, OmeB, was identified as the potential proton-conducting part of the OHR complex. Several highly conserved protonatable amino acids in combination with water-filled pores were proposed, enabling the extrusion of protons through the membrane. AlphaFold2 was used to construct an in-silico three-dimensional structure of the entire stand-alone OHR supercomplex, in agreement with experimental knowledge. In this model, one proton path identified in OmeB extends through the RDase, which catalyses the transfer of protons and electrons to the reduced halogenated substrate. This suggests that the RDase and its substrate in the respiration of CBDB1 replace the quinone function. In order to establish a method capable of purifying the oxygen-sensitive, membrane-bound OHR protein complex, various purification strategies were tested. A minimum of two steps consisting of anionexchange chromatography and size-exclusion chromatography or density gradient ultracentrifugation was found to be sufficient to isolate a 300 kDa and a 480 kDa protein complex. The RDase activity was retained in these complexes. Shotgun proteomics of the liquid fractions confirmed the presence of all seven expected subunits of the OHR complex. However, the detailed protein composition of bands excised from the CN-PAGE still needs to be determined by mass spectrometry. The experimental results show a very straightforward mechanism by which D. mccartyi couples proton extrusion through the membrane to substrate reduction and thus energy conservation without quinones, cytochromes or proton pumps. It was also shown that an oxygen-sensitive, membrane-bound OHR protein complex can be purified in its native form from very little biomass, enabling the establishment of an upscale approach for cryogenic electron microscopy (cryo-EM). These results could have relevance for other microbial processes and biotechnology applications.
Nadine Hellmold (Wed,) studied this question.