Protein Interactions in Rhodopseudomonas palustris TIE-1 Reveal the Molecular Basis for Resilient Photoferrotrophic Iron Oxidation.

Rhodopseudomonas palustris is an alphaproteobacterium with impressive metabolic versatility, capable of oxidizing ferrous iron to fix carbon dioxide using light energy. Photoferrotrophic iron oxidation is one of the most ancient metabolisms, sustained by the pio operon coding for three proteins: PioB and PioA, which form an outer-membrane porin-cytochrome complex that oxidizes iron outside of the cell and transfers the electrons to the periplasmic high potential iron-sulfur protein (HIPIP) PioC, which delivers them to the light-harvesting reaction center (LH-RC). Previous studies have shown that PioA deletion is the most detrimental for iron oxidation, while, the deletion of PioC resulted in only a partial loss. The expression of another periplasmic HiPIP, designated Rpal_4085, is strongly upregulated in photoferrotrophic conditions, making it a strong candidate for a PioC substitute. However, it is unable to reduce the LH-RC. In this work we used NMR spectroscopy to map the interactions between PioC, PioA, and the LH-RC, identifying the key amino acid residues involved. We also observed that PioA directly reduces the LH-RC, and this is the most likely substitute upon PioC deletion. By contrast, Rpal_4085 demontrated significant electronic and structural differences from PioC. These differences likely explain its inability to reduce the LH-RC and highlight its distinct functional role. Overall, this work reveals the functional resilience of the pio operon pathway and further highlights the use of paramagnetic NMR for understanding key biological processes.

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.

Characterization of the periplasmic redox network that sustains the versatile anaerobic metabolism of Shewanella oneidensis MR-1.

The versatile anaerobic metabolism of the Gram-negative bacterium Shewanella oneidensis MR-1 (SOMR-1) relies on a multitude of redox proteins found in its periplasm. Most are multiheme cytochromes that carry electrons to terminal reductases of insoluble electron acceptors located at the cell surface, or bona fide terminal reductases of soluble electron acceptors. In this study, the interaction network of several multiheme cytochromes was explored by a combination of NMR spectroscopy, activity assays followed by UV-visible spectroscopy and comparison of surface electrostatic potentials. From these data the small tetraheme cytochrome (STC) emerges as the main periplasmic redox shuttle in SOMR-1. It accepts electrons from CymA and distributes them to a number of terminal oxidoreductases involved in the respiration of various compounds. STC is also involved in the electron transfer pathway to reduce nitrite by interaction with the octaheme tetrathionate reductase (OTR), but not with cytochrome c nitrite reductase (ccNiR). In the main pathway leading the metal respiration STC pairs with flavocytochrome c (FccA), the other major periplasmic cytochrome, which provides redundancy in this important pathway. The data reveals that the two proteins compete for the binding site at the surface of MtrA, the decaheme cytochrome inserted on the periplasmic side of the MtrCAB-OmcA outer-membrane complex. However, this is not observed for the MtrA homologues. Indeed, neither STC nor FccA interact with MtrD, the best replacement for MtrA, and only STC is able to interact with the decaheme cytochrome DmsE of the outer-membrane complex DmsEFABGH. Overall, these results shown that STC plays a central role in the anaerobic respiratory metabolism of SOMR-1. Nonetheless, the trans-periplasmic electron transfer chain is functionally resilient as a consequence of redundancies that arise from the presence of alternative pathways that bypass/compete with STC.

Exploration of the 'cytochromome' of Desulfuromonas acetoxidans, a marine bacterium capable of powering microbial fuel cells.

Recent progress in bacterial genomic analysis has revealed a vast number of genes that encode c-type cytochromes that contain multiple heme cofactors. This high number of multiheme cytochromes in several bacteria has been correlated with their great respiratory flexibility, and in what concerns biotechnological applications, has been correlated with electricity production in Microbial Fuel Cells. Desulfuromonas acetoxidans, a member of the Geobactereaceae family, is one of these organisms for which the genome was recently made available, coding for 47 putative multiheme cytochromes. The growth of D. acetoxidans in different media allowed the identification of the cytochromes dominant in each condition. The triheme cytochrome c(7) is always present suggesting a key role in the bioenergetic metabolism of this organism, and a dodecaheme cytochrome of low homology with other proteins in the databases was also isolated. Different cytochromes are found for different growth conditions showing that their roles can be assigned to specific bioenergetic electron transfer routes.