In the context of the development of bioprocesses for the production of biofuels and bulk chemicals, microbial cells are rationally engineered to produce such molecules at high yield and titres in order to develop new biological methods that satisfy economic constraints. The redox and energetic balances of such strains play crucial roles in performance. Processes performed in strict anaerobes have a limited amount of energy available compared to that in aerobic organisms. This energy is obtained through fermentation and/or ion gradient-driven phosphorylation. Such anaerobic organisms have developed energy conservation mechanisms to increase ATP yields. This paper presents the properties of one of these mechanisms catalysed by the Rnf complex, an ion-translocating membrane complex with a ferredoxin NAD+ oxidoreductase activity. The Rnf complex performs the transfer of electrons from reduced ferredoxin to NAD+ coupled with an ion-motive transport. Ferredoxin is a common electron carrier for anaerobic bacteria and, with NAD+, is involved in several pathways of interest for the production of biofuels. This complex was first identified in Rhodobacter capsulatus and found to be involved in nitrogen fixation. It was then found to be involved in energy conservation in multiple anaerobic organisms, from acetobacteria such as Acetobacterium woodii to sulfate-reducing bacteria such as Desulfovibrio alaskensis and autotrophic bacteria such as Clostridium ljungdahlii and Clostridium aceticum. The Rnf complex triggers two types of ion transports: it can be either a sodium or a proton transporter. Both of these transports create a gradient of ions, generating a membrane potential that is then used by ATPase to produce ATP and thus serving as an energy conservation mechanism. In this review, the available information on the Rnf complex from genetic organization up to its in vivo and in vitro activities in several microorganisms is summarized, with a special focus on the proton-motive Rnf complex.

The molecular entity that catalyzes ferredoxin (Fd)-dependent cyclic electron flow around photosystem I (PSI) (Fd-CEF) remains unknown. To elucidate the in vivo molecular mechanism of Fd-CEF, the evaluation of Fd reduction‒oxidation kinetics is a good indicator of Fd-CEF activity. Recent research showed that the expression of Fd-CEF activity requires the oxidation of plastoquinone, and furthermore, chloroplast NAD(P)H dehydrogenase does not catalyze Fd-CEF in Arabidopsis thaliana. In the present research, the effect of reduced Fd on Fd-CEF activity was analyzed by comparing wild-type and pgr5-deficient mutants (pgr5hope1). PGR5 has been proposed to be the mediator of Fd-CEF, and pgr5hope1 showed the same CO2 assimilation rate and the same reduction‒oxidation level of PQ as WT, but P700 oxidation with Fd was highly reduced, unlike WT. As expected, the activity of Fd-CEF was enhanced in pgr5hope1 compared to the wild type, and its activity was also enhanced with the oxidation of PQ by the increase in the CO2 assimilation rate. The present in vivo research clearly shows that the expression of Fd-CEF activity requires reduced Fd in addition to oxidized PQ, and PGR5 does not catalyze Fd-CEF.

Simple Summary: The distribution of typical bacterial redox enzymes such as pyruvate-ferredoxin oxidoreductase (PFOR) in protozoa remains interestingly puzzling. Previous studies have demonstrated diverse cellular localizations of PFOR in some amitochondriate anaerobic protozoa. PFOR is of particular pharmacological importance because it catalyzes the reductive bio-activation of nitro-based prodrugs to cytotoxic radical metabolites. Metronidazole was developed primarily as an antiprotozoal agent against infections caused by Trichomonas vaginalis. However, its antimicrobial spectrum was subsequently expanded to cover anaerobic bacterial infections. It has been shown that mutations in the genes encoding PFOR result in inherent resistance of PFOR-possessing anaerobic protozoa and bacteria to nitro-based prodrugs. Deciphering the evolutionary history of PFOR is crucial in deepening our understanding of the evolution of anaerobic pathogens and unfold new approaches for drug discovery and targeting in pathogen chemotherapy. Abstract: The present frontrunners in the chemotherapy of infections caused by protozoa are nitro-based prodrugs that are selectively activated by PFOR-mediated redox reactions. This study seeks to analyze the distribution of PFOR in selected protozoa and bacteria by applying comparative bioinformatics approaches to test the hypothesis that PFOR was most likely acquired through horizontal gene transfer (HGT) from bacteria. Furthermore, to identify other genes that were putatively acquired using proteome-wide and gene expression analyses. A plausible explanation for the patchy occurrence of PFOR in protozoa is based on the hypothesis that bacteria are potential sources of genes that enhance optimal adaptation of protozoa in hostile environments. The exclusively expressed proteins obtained from Entamoeba histolytica and the putative gene donor, Desulfovibrio vulgaris, showed an over-expression of eleven genes involved in intermediary metabolism. If these results can be reproduced in other PFOR-possessing protozoa, it would provide a more validated evidence to support the horizontal transfer of pfor from bacteria.

Direct electron transfer (DET)-type bioelectrocatalysis, which couples electrode reactions and catalytic functions of redox enzymes without any redox mediator, is one of the most intriguing subjects studied over the past decades in the field of bioelectrochemistry. In order to realize the DET-type bioelectrocatalysis and to improve the performance, nanostructures of the electrode surface have to be carefully tuned for each enzyme. In addition, enzymes can also be tuned by protein engineering approach for the DET-type reaction. This review summarizes the resent progresses in this field of the research, while taking into consideration of the importance of nanostructure of electrodes as well as redox enzymes. Described are basic concepts and theoretical aspects of DET-type bioelectrocatalysis, significance of nanostructures as scaffolds for DET-type reactions, protein engineering approached for DET-type reactions, and concepts and facts of bidirectional DET-type reactions, from a cross-disciplinary viewpoint.