Abstract
Dehalococcoides mccartyi strain CBDB1 is a slow growing strictly anaerobic microorganism dependent on halogenated compounds as terminal electron acceptor for anaerobic respiration. Indications have been described that the membrane-bound proteinaceous organohalide respiration complex of strain CBDB1 is functional without quinone-mediated electron transfer. We here study this multi-subunit protein complex in depth in regard to participating protein subunits and interactions between the subunits using blue native gel electrophoresis coupled to mass spectrometric label-free protein quantification. Applying three different solubilization modes to detach the respiration complex from the membrane we describe different solubilization snapshots of the organohalide respiration complex. The results demonstrate the existence of a two-subunit hydrogenase module loosely binding to the rest of the complex, tight binding of the subunit HupX to OmeA and OmeB, predicted to be the two subunits of a molybdopterin-binding redox subcomplex, to form a second module, and the presence of two distinct reductive dehalogenase module variants with different sizes. In our data we obtained biochemical evidence for the specificity between a reductive dehalogenase RdhA (CbdbA80) and its membrane anchor protein RdhB (CbdbB3). We also observed weak interactions between the reductive dehalogenase and the hydrogenase module suggesting a not yet recognized contact surface between these two modules. Especially an interaction between the two integral membrane subunits OmeB and RdhB seems to promote the integrity of the complex. With the different solubilization strengths we observe successive disintegration of the complex into its subunits. The observed architecture would allow the association of different reductive dehalogenase modules RdhA/RdhB with the other two protein complex modules when the strain is growing on different electron acceptors. In the search for other respiratory complexes in strain CBDB1 the remarkable result is not the detection of a standard ATPase but the absence of any other abundant membrane complex although an 11-subunit version of complex I (Nuo) is encoded in the genome.
Highlights
Organohalides are used in many different products such as pesticides, biocides, pharmaceuticals, plasticizers, personal care articles and flame retardants but are produced by natural processes
One particular process favored for bioremediation of organohalides in sediments, soils or groundwater is organohalide respiration catalyzed by organohalide-respiring bacteria (Steffan and Schaefer, 2016)
The reaction would in some way resemble the Abbreviations: DDM, N-dodecyl-ß-D-maltoside; BN-PAGE, blue native polyacrylamide gel electrophoresis; OHR, Organohalide respiration
Summary
Organohalides are used in many different products such as pesticides, biocides, pharmaceuticals, plasticizers, personal care articles and flame retardants but are produced by natural processes. Organohalide-respiring bacteria use the halogenated compounds as a terminal electron acceptor in an anaerobic respiration and gain energy for growth from this process. The current hypothesis is that the OHR complex represents a fully functional stand-alone respiratory chain, obviating quinone or cytochrome involvement (Kublik et al, 2016; Hartwig et al, 2017). The other proposed subunits in the OHR complex are RdhB, which is the putative membrane integral RdhA anchoring protein, the organohalide respiration involved molybdoenzyme (OmeA), its putative membraneintegrated anchor OmeB, HupX, a protein with four predicted iron-sulfur clusters and a hydrogen uptake hydrogenase with its [NiFe] large subunit (HupL) and iron-sulfur containing small subunit (HupS) (Figure 1). The current mechanistic model hypothesizes that electrons are fed into the complex via HupL, and transferred via ironsulfur clusters in HupS, HupX and OmeA to the RdhA which reduces organohalides. The reaction would in some way resemble the Abbreviations: DDM, N-dodecyl-ß-D-maltoside; BN-PAGE, blue native polyacrylamide gel electrophoresis; OHR, Organohalide respiration
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