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Figure 3.4 Schematic of multiheme cytochromes involved in transporting electrons from the organic substrate within the cell to the surface of an iron oxide mineral in (A) Shewanella and (B) Geobacter, adapted from Shi et al. (2007), Santos et al. (2015), and Shi et al. (2016). (A) When MR‐1 oxidizes a carbon substrate such as lactate by a dehydrogenase, electrons from this reaction are transported to a quinone/quinol pool. Electrons are passed onto cytoplasmic and periplasmic multiheme cytochromes and to the outermembrane multiheme cytochromes. These cytochromes can then reduce the iron oxide surface directly or further pass electrons via an extracellular transport system. (B) In Geobacter sulfurreducens, electrons derived from the oxidation of a carbon substrate by a dehydrogenase can also be passed onto a quinone/quinol pool and then onto cytoplasmic (e.g. ImH, CbcL) and periplasmic multiheme cytochromes (e.g. PpcA), from which electrons can be delivered to outer membrane multiheme cytochromes (e.g. OmbB and OmbC). Alternatively, electrons can be transferred to outermembrane cytochromes (e.g. OmcE and OmcS), which are associated with the pili, further passing the electrons to the iron oxide. The pili themselves are also capable of extracellular electron transfer.

What is best understood about extracellular electron transport is the general pathway of the electron from the cytoplasm to ferric iron (Figure 3.4). The passage of electrons to iron, residing on the cell exterior, happens by way of enzymes and molecules that traverse the inner and outer membranes. In the bacterial strains Shewanella MR‐1 and ANA‐3, a series of multi‐heme cytochrome proteins can shuttle electrons from inside the cytoplasmic membrane to the outer membrane (El‐Naggar et al. 2010; Clarke et al. 2011; Reyes et al. 2012; Watanabe et al. 2017). During iron reduction (e.g. ferrihydrite or iron(III)‐citrate) electrons are donated from a reduced substrate in the cytoplasm (e.g. lactate) to a dehydrogenase in the inner membrane (Burns 2010). Proton translocation from the cytoplasm to the periplasm is presumed to occur as electrons proceed via a quinone pool (quinones and quinols embedded in the membrane) to the periplasmic cytochrome CymA (Shi et al. 2016). From CymA, electrons are passed to the periplasmic cytochromes Fcc3 and STC (Shi et al. 2016). These cytochromes in turn transfer electrons to MtrA, a decaheme cytochrome in the periplasm (Courselle et al. 2010), and MtrA transfers the electrons to the outer membrane cytochromes MtrC and OmcA via the porin protein MtrB (Shi et al. 2016).

      Hypotheses exist regarding how electrons could be transferred from certain hemes of the outer membrane cytochrome MtrF in MR‐1 (Clarke et al. 2011) to iron oxide (see below), but whether the electron transfer per se is specific to particular electron acceptors remains unclear. To date, there is only one study, involving Shewanella oneidensis MR‐1 that has provided protein‐crystallographic evidence of how iron as iron(III)‐citrate and iron(III)‐NTA bind to specific sites of the outer membrane cytochrome UndA before reduction (Edwards et al. 2012). Single frozen crystals of UndA, soaked in chelated iron, were analysed by synchrotron radiation. While binding sites near heme 7 of the undecaheme cytochrome UndA of MR‐1 appear to specifically bind chelated iron(III), they do not appear to differentiate between the type of chelator, e.g. NTA or citrate (Edwards et al. 2012). Thus, sites near heme 7 could in general bind different types of chelated iron(III). These results imply that at least for this cytochrome there are specific sites for soluble iron reduction.

      Geobacter sulfurreducens possesses at least 30 outer membrane cytochromes (Costa et al. 2018), although they are not structurally characterized to the extent of those of MR‐1, and it is not known which hemes interact with different electron acceptors. Otero et al. (2018) have shown that multiple pathways of iron‐oxide and iron(III)‐citrate reduction are possible in G. sulfurreducens involving different combinations of cytochromes, supporting results from earlier studies. One proposed pathway for the reduction of iron oxides involves electron transfer from a quinone pool to multiheme cytochromes ImcH (Levar et al. 2014) and CbcL (Zacharoff et al. 2015) in the inner membrane. These in turn could transfer electrons to the periplasmic cytochrome PpcA (Lloyd et al. 2003) and from PpcA to the outer membrane cytochromes OmcBC via the porin proteins OmaBC (Shi et al. 2016). Other outer membrane cytochromes OmcE and OmcS were shown to be important for reduction of iron oxides but not of iron(III)‐citrate (Mehta et al. 2005).

      3.3.2. Microbial Strategies to Reduce Solid Iron Phases

      In addition to the fact that the electrons have to be channeled from the substrate to the electron acceptor, it is particularly challenging for microorganisms that iron is usually not dissolved under marine, circumneutral pH conditions. Despite these challenges, several strategies still allow bacteria to deliver electrons to a solid‐phase electron acceptor. Biochemical methods have allowed for whole cell interactions with metal oxides (Lower et al. 2007) and electrodes (Coursolle et al. 2010) to be determined and abiotic (Lower et al. 2007, 2008; Clarke et al. 2011; Breuer et al. 2015) and biotic (Lower et al. 2007) binding of specific multiheme cytochromes with different iron oxide mineral surfaces to be resolved. However, the mechanism of electron transport from the hemes to these solid‐phase electron acceptors is still unknown.

      Many studies of electron transfer from MR‐1 cell suspensions to the surface of electrodes in microbial fuel cells and in cyclic voltammetry have been made using different types of electrodes including glassy carbon (Kim et al. 1999, p. 128; Kim et al. 2002), graphite (Bretschger et al. 2007; Wang et al. 2011), and hematite (Meitl et al. 2009). In all cases under specific experimental conditions with specific redox potentials, the cells have been able to deliver electrons to these various types of electrodes. Thus, it is irrelevant that the electron acceptor is iron, as electrons would be equally transported to any electrode with a particular electron potential. What matters is that the microorganism has the molecular pathway to transfer electrons from a substrate in the cell interior to an extracellular electron acceptor.

      Theoretically, the bacteria could be growing directly on the surface of the iron mineral, which is indeed observed in laboratory experiments (Jeong et al. 2006). However, this is not always possible in natural sediments. Organisms have developed strategies to transport electrons to the mineral surface, either via secreted organic molecules that act as electron shuttles, such as flavins, or through extensions of the outer membrane, such as nanowires, capable of transporting electrons. Alternatively, there are also strategies to transport Fe³⁺ ions from the mineral to the cell, using chelators (i.e. siderophores). In any case, the communities have to be highly organized, as otherwise the energetic costs of manufacturing such structures

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