A New Paradigm of Multiheme Cytochrome Evolution by Grafting and Pruning Protein Modules.

Multiheme cytochromes play key roles in diverse biogeochemical cycles, but understanding the origin and evolution of these proteins is a challenge due to their ancient origin and complex structure. Up until now, the evolution of multiheme cytochromes composed by multiple redox modules in a single polypeptide chain was proposed to occur by gene fusion events. In this context, the pentaheme nitrite reductase NrfA and the tetraheme cytochrome c554 were previously proposed to be at the origin of the extant octa- and nonaheme cytochrome c involved in metabolic pathways that contribute to the nitrogen, sulfur, and iron biogeochemical cycles by a gene fusion event. Here, we combine structural and character-based phylogenetic analysis with an unbiased root placement method to refine the evolutionary relationships between these multiheme cytochromes. The evidence show that NrfA and cytochrome c554 belong to different clades, which suggests that these two multiheme cytochromes are products of truncation of ancestral octaheme cytochromes related to extant octaheme nitrite reductase and MccA, respectively. From our phylogenetic analysis, the last common ancestor is predicted to be an octaheme cytochrome with nitrite reduction ability. Evolution from this octaheme framework led to the great diversity of extant multiheme cytochromes analyzed here by pruning and grafting of protein modules and hemes. By shedding light into the evolution of multiheme cytochromes that intervene in different biogeochemical cycles, this work contributes to our understanding about the interplay between biology and geochemistry across large time scales in the history of Earth.

Investigation of the Molecular Mechanisms of the Eukaryotic Cytochrome-c Maturation System.

Cytochromes-c are ubiquitous heme proteins with enormous impact at the cellular level, being key players in metabolic processes such as electron transfer chains and apoptosis. The assembly of these proteins requires maturation systems that catalyse the formation of the covalent thioether bond between two cysteine residues and the vinyl groups of the heme. System III is the maturation system present in Eukaryotes, designated CcHL or HCCS. This System requires a specific amino acid sequence in the apocytochrome to be recognized as a substrate and for heme insertion. To explore the recognition mechanisms of CcHL, the bacterial tetraheme cytochrome STC from Shewanella oneidensis MR-1, which is not a native substrate for System III, was mutated to be identified as a substrate. The results obtained show that it is possible to convert a bacterial cytochrome as a substrate by CcHL, but the presence of the recognition sequence is not the only factor that induces the maturation of a holocytochrome by System III. The location of this sequence in the polypeptide also plays a role in the maturation of the c-type cytochrome. Furthermore, CcHL appears to be able to catalyse the binding of only one heme per polypeptide chain, being unable to assemble multiheme cytochromes c, in contrast with bacterial maturation systems.

Electron transfer in Gram-positive bacteria: enhancement strategies for bioelectrochemical applications.

To this day, bioelectrochemical systems are still perceived as one of the rising technologies due to their versatile applications in electricity production, bioremediation, biosensors, and production of value-added products. While the majority of bioelectrochemical applications utilize Gram-negative bacteria, Gram-positive bacteria has not received sufficient attention. The lack of adequate knowledge about their electron transfer pathways along with the presence of a thick non-conductive cell wall are among the reasons behind their limited use. In this review, the electroactivity of Gram-positive bacteria will be covered describing the different pathways of electron transfer among different electroactive Gram-positive strains. Special emphasis will be given to the role of multiheme cytochromes, quorum sensing molecules, peptide-based signalling, and pili in the extracellular electron transfer. This review will also provide an overview of possible approaches for enhancement strategies of electron transfer such as enhancing biofilm formation, biocomposites and cell perforation. Understanding the fundamentals is critical for improving the use of Gram-positive bacteria in bioelectrochemical systems and may lead to the discovery of new applications.

Electrografted anthraquinone to monitor pH at the biofilm-anode interface in a wastewater microbial fuel cell.

Electrografted anthraquinone on graphite was used as a probe to monitor the pH change at the biofilm-electrode interface at the anode of a microbial fuel cell inoculated with wastewater. The grafting procedure was optimized so that the pH-dependent electrochemical response of the grafted quinone did not overlay with that of the electroactive biofilm. The variation of the formal potential of the grafted quinone as a function of pH was linear over the pH range 1-10 with a slope of - 64 mV. This allowed to monitor the interfacial pH change over three weeks of biofilm colonization of the electrode. During that time the interfacial pH decreased from neutrality to 5.3 while the anolyte only acidified down to pH 6.2. This finding is relevant as local pH change usually leads to alterations of the bioenergetics process of microbial communities and hence on the performance of bioelectrochemical devices.

Crossing the Wall: Characterization of the Multiheme Cytochromes Involved in the Extracellular Electron Transfer Pathway of Thermincola ferriacetica.

Bioelectrochemical systems (BES) are emerging as a suite of versatile sustainable technologies to produce electricity and added-value compounds from renewable and carbon-neutral sources using electroactive organisms. The incomplete knowledge on the molecular processes that allow electroactive organisms to exchange electrons with electrodes has prevented their real-world implementation. In this manuscript we investigate the extracellular electron transfer processes performed by the thermophilic Gram-positive bacteria belonging to the Thermincola genus, which were found to produce higher levels of current and tolerate higher temperatures in BES than mesophilic Gram-negative bacteria. In our study, three multiheme c-type cytochromes, Tfer_0070, Tfer_0075, and Tfer_1887, proposed to be involved in the extracellular electron transfer pathway of T. ferriacetica, were cloned and over-expressed in E. coli. Tfer_0070 (ImdcA) and Tfer_1887 (PdcA) were purified and biochemically characterized. The electrochemical characterization of these proteins supports a pathway of extracellular electron transfer via these two proteins. By contrast, Tfer_0075 (CwcA) could not be stabilized in solution, in agreement with its proposed insertion in the peptidoglycan wall. However, based on the homology with the outer-membrane cytochrome OmcS, a structural model for CwcA was developed, providing a molecular perspective into the mechanisms of electron transfer across the peptidoglycan layer in Thermincola.

Electron transfer process in microbial electrochemical technologies: The role of cell-surface exposed conductive proteins.

Electroactive microorganisms have attracted significant interest for the development of novel biotechnological systems of low ecological footprint. These can be used for the sustainable production of energy, bioremediation of metal-contaminated environments and production of added-value products. Currently, almost 100 microorganisms from the Bacterial and Archaeal domains are considered electroactive, given their ability to efficiently interact with electrodes in microbial electrochemical technologies. Cell-surface exposed conductive proteins are key players in the electron transfer between cells and electrodes. Interestingly, it seems that among the electroactive organisms identified so far, these cell-surface proteins fall into one of four groups. In this review, the different types of cell-surface conductive proteins found in electroactive organisms will be overviewed, focusing on their structural and functional properties.

Modulation of the reactivity of multiheme cytochromes by site-directed mutagenesis: moving towards the optimization of microbial electrochemical technologies.

Dissimilatory metal-reducing bacteria perform extracellular electron transfer, a metabolic trait that is at the core of a wide range of biotechnological applications. To better understand how these microorganisms transfer electrons from their metabolism to an extracellular electron acceptor, it is necessary to characterize in detail the key players in this process, the multiheme c-type cytochromes. Shewanella oneidensis MR-1 is a model organism for studying extracellular electron transfer, where the heme protein referred to as small tetraheme cytochrome is one of the most abundant multiheme cytochromes found in the periplasmic space of this bacterium. The small tetraheme cytochrome is responsible for the delivery of electrons to the porin-cytochrome supercomplexes that permeate the outer-membrane and reduce metallic minerals or electrodes. In this work, well-established thermodynamic and kinetic models that discriminate the electron transfer activity of the four individual hemes were employed to characterize a set of single amino-acid mutants of the small tetraheme cytochrome and their interaction with small inorganic electron donors and acceptors. The results show that electrostatics play an important role in the reactivity of the small tetraheme cytochrome with small inorganic electron partners, in particularly in the kinetics of the electron transfer processes. This thorough exploration using site-directed mutants provides key mechanistic insights to guide the rational manipulation of the proteins that are key players in extracellular electron transfer processes, towards the improvement of microbial electrochemical applications using dissimilatory metal-reducing bacteria.