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

Abstract

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.

Electrochemical Microwell Plate to Study Electroactive Microorganisms in Parallel and Real-Time

“…This will open the door to perform microbial resource mining in habitats that are already well known for harbouring EAM like wastewater and soil ( Koch and Harnisch 2016a ; Logan et al, 2019 ) but also recently discovered ones like the oral ( Naradasu et al, 2020 ) or gut ( Tahernia et al, 2020b ; Rago et al, 2021 ) microbiome and especially to explore new habitats as resources. Further, we foresee that already exploited EAM, for instance, for microbial electrosynthesis of chemical building blocks ( Mayr et al, 2019 ; Wu et al, 2019 ), can be further improved using concepts and tools that are well established (for non-electrochemical means) like site directed mutagenesis, CRISPR-CAS, and techniques beyond in high-throughput ( Alves et al, 2017 ; Fan et al, 2020 ). Even longer and more complex measurements will become a possibility by integrating microfluidics ( Yoon et al, 2018 ; Yates et al, 2021 ; Li et al, 2011 ), e.g., to replenish culture media or to enable complex co-cultivation experiments.…”

Optimizing Electroactive Organisms: The Effect of Orthologous Proteins

“…The elucidation of the molecular basis of the electron transfer processes performed by multiheme c-type cytochromes is crucial for the efficient rational manipulation of electroactive organisms toward specific properties and toward their practical implementation in MET (Fonseca et al, 2009;Alves et al, 2017;Neto et al, 2017). As one of the most abundant protein in the periplasmic space of Shewanella, STC plays an important role in extracellular electron transfer (Gordon et al, 2000;Sturm et al, 2015), being crucial together with FccA for the electron transfer across the periplasm (Fonseca et al, 2013;Alves et al, 2015;Sturm et al, 2015).…”

“…For protein film voltammetry (PFV) experiments pure SaSTC (in 20 mM phosphate buffer with 100 mM KCl at pH 7.6) was mixed with poly-L-lysine solution (0.1% in H 2 0 from SIGMA) in a mixture of 1:1 to facilitate the adsorption of the protein to the electrode (Alves et al, 2017). A small volume of this mixture was deposited onto pyrolytic graphite edge (PGE) electrode and was left to dry.…”

“…In recent years, significant efforts have been made to characterize the molecular details of the electron transfer processes performed by these proteins so rational manipulation can be used to enhance electron transfer rates (Fonseca et al, 2009;Alves et al, 2017;Neto et al, 2017). However, the molecular factors that underpin the electron transfer rates in this redox chain are still unclear.…”

“…However, the molecular factors that underpin the electron transfer rates in this redox chain are still unclear. In vitro studies of site directed mutants of these multiheme cytochromes have shown that electrostatic and thermodynamics are important factors in controlling electron transfer (Alves et al, 2017;Neto et al, 2017). However, it is still not known if changes in these parameters affect electron transfer in living cells, since cellular milieu influences protein folding, stability and the reactivity of the proteins (Kuznetsova et al, 2014;Gnutt and Ebbinghaus, 2016).…”