The Membrane-Bound C Subunit of Reductive Dehalogenases: Topology Analysis and Reconstitution of the FMN-Binding Domain of PceC

Abstract

Organohalide respiration (OHR) is the energy metabolism of anaerobic bacteria able to use halogenated organic compounds as terminal electron acceptors. While the terminal enzymes in OHR, so-called reductive dehalogenases, are well-characterized, the identity of proteins potentially involved in electron transfer to the terminal enzymes remains elusive. Among the accessory genes identified in OHR gene clusters, the C subunit (rdhC) could well code for the missing redox protein between the quinol pool and the reductive dehalogenase, although it was initially proposed to act as transcriptional regulator. RdhC sequences are characterized by the presence of multiple transmembrane segments, a flavin mononucleotide (FMN) binding motif and two conserved CX3CP motifs. Based on these features, we propose a curated selection of RdhC proteins identified in general sequence databases. Beside the Firmicutes from which RdhC sequences were initially identified, the identified sequences belong to three additional phyla, the Chloroflexi, the Proteobacteria, and the Bacteriodetes. The diversity of RdhC sequences mostly respects the phylogenetic distribution, suggesting that rdhC genes emerged relatively early in the evolution of the OHR metabolism. PceC, the C subunit of the tetrachloroethene (PCE) reductive dehalogenase is encoded by the conserved pceABCT gene cluster identified in Dehalobacter restrictus PER-K23 and in several strains of Desulfitobacterium hafniense. Surfaceome analysis of D. restrictus cells confirmed the predicted topology of the FMN-binding domain (FBD) of PceC that is the exocytoplasmic face of the membrane. Starting from inclusion bodies of a recombinant FBD protein, strategies for successful assembly of the FMN cofactor and refolding were achieved with the use of the flavin-trafficking protein from D. hafniense TCE1. Mass spectrometry analysis and site-directed mutagenesis of rFBD revealed that threonine-168 of PceC is binding FMN covalently. Our results suggest that PceC, and more generally RdhC proteins, may play a role in electron transfer in the metabolism of OHR.

Organohalide-respiringDesulfolunaspecies isolated from marine environments

“…3), a homolog of proteins previously proposed to function as transcriptional regulator for rdhAB gene expression in Desulfitobacterium dehalogenans [73]. However, a recent study on PceC from Dehalobacter restrictus proposed a possible role for RdhC in electron transfer from menaquinones to PceA via its exocytoplasmically-facing flavin mononucleotide (FMN) cofactor [16]. RdhC in Desulfoluna strains also showed the conserved FMN binding motif (in particular the fully conserved threonine residue) and two CX 3 CP motifs predicted to have a role in electron transfer [16] (Fig.…”

“…The electron transport chain from electron donors to RDases has been classified into quinone-dependent (that rely on menaquinones as electron shuttles between electron donors and RDases) and quinone-independent pathways [9, 10, 14]. Recent studies suggested that RdhC may serve as electron carrier during OHR in Firmicutes [15, 16].…”

Impact of iron reduction on the metabolism of Clostridium acetobutylicum

“…We hypothesize that FAD is hydrolyzed extracellularly to FMN under iron-reducing conditions and covalently bound to a membrane enzyme with a potential role in iron reduction, since the disappearance of FAD is only observed in the iron-reducing cultures. The hydrolysis of FAD to FMN with incorporation into a membrane protein was recently proposed for other Gram-positive bacteria (Buttet et al, 2018). Additionally, a flavin-based extracellular electron transfer mechanism was proposed for Gram-positive bacteria, in which FAD is converted to FMN, which is bound to a predicted extracellular electron transfer lipoprotein (Light et al, 2018).…”

Organohalide-respiring Desulfoluna species isolated from marine environments

“…Qrc was detected in the proteome of DBB cells grown under both LS and LSD conditions (Table S5), whereas the TpIc3 was not identified in the Desulfoluna genomes. Instead of TpIc3, strain DBB could use RdhC1, a homolog to PceC of Dehalobacter restrictus that was proposed to mediate electron transfer from menaquinol to PceA via its exocytoplasmic-facing flavin mononucleotide (FMN) cofactor [16]. Similar to D. restrictus, the RdhC1 of strain DBB contains a conserved FMN binding motif (in particular the fully conserved threonine residue) and two CX 3 CP motifs predicted to have a role in electron transfer [16] (Fig.…”

“…The electron transport chain from electron donors to RDases has been classified into quinone-dependent (relying on menaquinones as electron shuttles between electron donors and RDases) and quinone-independent pathways [9,10,14]. Recent studies suggested that RdhC may serve as electron carrier during OHR in Firmicutes [15,16].…”