Extracellular electron transfer (EET) is a general mechanism by which microorganisms transfer electrons across their cell membrane as part of their metabolic pathways. EET describes the processes by which organisms pass electrons between the intracellular and extracellular environments. EET plays a crucial role in biogeochemical cycling, particularly of iron and manganese in marine and terrestrial ecosystems (Shi et al., 2016; Tremblay et al., 2017). By modulating redox gradients, EET-active microbes influence sediment chemistry and microbial community structures, impacting carbon mineralization and anaerobic respiration. Beyond metal cycling, EET enables direct interspecies electron transfer (DIET), where electron-donating bacteria such as Geobacter spp. transfer electrons to syntrophic partners like methanogenic archaea, (Gao and Lu, 2021), or between Geobacter species to enable oxidation of diverse organic compounds (Summers et al., 2010). In addition to these roles, EET contributes significantly to plant and animal ecosystems by enhancing nutrient uptake in plant rhizospheres and influencing gut redox balance, host immunity, and pathogen colonization in animals (Stevens and Marco, 2023).

EET underpins biotechnological applications, including bioremediation, microbial fuel cells (MFCs), microbial electrosynthesis, metal recovery, and electric syntrophy. (Kato, 2015). In bioremediation, microbial EET reduces toxic metals such as Cr (VI) and U (VI) to insoluble forms, limiting their bioavailability and environmental impact (Wang and Shen, 1995; Lovley and Coates, 1997). MFCs exploit EET to convert organic substrates into electrical energy, enabling sustainable wastewater treatment and bioelectricity generation (Logan, 2009). Microbial electrosynthesis uses EET for CO₂ fixation into value-added chemicals like acetate and biofuels (Kato, 2015). Additionally, EET enhances anaerobic ammonium oxidation (anammox), offering new strategies for nitrogen removal in wastewater treatment (Zhen et al., 2024). Expanding our understanding of microbial EET mechanisms holds great potential for advancing energy, environmental, and industrial biotechnology.

This review highlights recent advances in understanding heme-mediated EET mechanisms which enable an environmentally and technologically important form of anaerobic respiration. Using EET, anaerobic organisms can access extracellular electron acceptors, including soluble metal species, solid state minerals, or synthetic electrodes, as part of energy generating metabolic processes (Shi et al., 2016). EET occurs through proteins in direct contact with electron acceptors or donors, or via diffusible redox shuttles. We will survey the present understanding of the role of heme proteins, called cytochromes, in direct EET pathways employed primarily by the model organism, Geobacter sulfurreducens. These pathways encompass electron transfer from intracellular donors (e.g., menaquinone) to inner membrane, periplasmic, and outer membrane cytochromes and finally to extracellular electron acceptors.

Geobacter sulfurreducens is a highly adaptive organism capable of respiring anaerobically on a range of terminal electron acceptors, both soluble and insoluble, in liquid cultures, as well as on solid state mineral and electrode surfaces relevant to microbial electrochemical technologies. The discovery and study of G. sulfurreducens as a model organism for EET were shaped by early studies that established its genetic tractability and unique respiratory capabilities. The development of a genetic system for G. sulfurreducens allowed researchers to introduce foreign DNA, create targeted gene mutants, and manipulate and develop reporters of gene expression, all critical steps in unraveling its EET pathways (Coppi Maddalena V. et al., 2001). Its genetic tractability, combined with foundational work demonstrating the organism’s ability to use electrodes as electron acceptors, cemented its role in the study of electrical current generation in MFCs (Bond and Lovley, 2003). Unlike organisms such as S. oneidensis, extracellular reduction of Fe(III) oxides by G. sulfurreducens requires direct contact between the organism and the insoluble electron acceptor (Nevin and Lovley, 2000). Early studies of dissimilatory metal reduction proposed the involvement of cytochromes in EET by the related bacterium Geobacter metallireducens, (Gorby and Lovley, 1991), and later work confirmed their requirement for the reduction of soluble (Leang et al., 2003; Lloyd et al., 2003; Butler et al., 2004) and insoluble (Mehta T. et al., 2005) electron acceptors by G. sulfurreducens. These findings placed the role of multiheme c-type cytochromes (MHCs) in shuttling electrons across cell membranes and to remote extracellular electron acceptors into sharp focus for this field of research in the following decades.

Another defining feature of G. sulfurreducens is its ability to form conductive biofilms that enable long-range electron transport and support dense packing of whole cell catalysts on anodes for microbial fuel cells. These biofilms maintain efficient electron transfer throughout the community to support anaerobic respiration several cell layers away from those in direct contact with the electrodes (Gorby and Lovley, 1991; Reguera et al., 2006). G. sulfurreducens enables this mode of growth by secreting a conductive biofilm matrix through which electrons shed by constituent cells access the oxidative potential of the anode surface. While the identity of the conductive elements that comprise this matrix was hotly debated, (Bond et al., 2012; Snider et al., 2012; Yates et al., 2016), these are now widely accepted to be networks of MHCs (see Section 5.2 for more details).

Geobacter sulfurreducens has become a model organism for studying the role of hemes in microbial anaerobic respiration. MHCs play a critical role in its adaptation and survival. Based on a search of its annotated genome for c-type heme binding motifs (CXXCH), G. sulfurreducens encodes 132 putative c-type cytochromes, 78 of which are MHCs (≥2 CXXCH motifs per protein). MHCs represent more than an order of magnitude greater fraction of the G. sulfurreducens proteome as compared to model microbes Escherichia coli (bacterium), Saccharomyces cereviciae (fungi/yeast), and even humans (Homo sapiens) (Figure 1). It is genetically tractable, exhibits numerous respiratory pathways that transport electrons into the extracellular environment, can grow as planktonic and biofilm phenotypes, and has varied contributions of MHCs to facilitating these respiratory pathways. Consequently, it provides fertile ground for studying a range of heme mediated electron transport phenomenon in microbial biology.

In G. sulfurreducens, electrons are primarily generated through the complete oxidation of acetate via the tricarboxylic acid (TCA) cycle, producing NADH, NADPH, and reduced ferredoxin (Galushko and Schink, 2000; Mahadevan R. et al., 2006). Additional contributions can come from formate or hydrogen oxidation depending on growth conditions, but the TCA cycle remains the dominant source of reducing equivalents (Butler et al., 2006; Yang et al., 2010). Electrons derived from cytoplasmic metabolism, such as NADH oxidation, are transferred to the menaquinone (MK) pool via NADH dehydrogenase or alternative dehydrogenases, including formate and hydrogen dehydrogenases (Levar et al., 2017; Pimenta et al., 2023). NADH dehydrogenase (Complex I), for example, reduces menaquinone (MK) to menaquinol (MKH₂) and transports protons across the inner membrane to generate a proton motive force (PMF). This is one of the few energy-conserving steps in G. sulfurreducens, and its continued function depends on maintaining an oxidized quinone pool (Meng et al., 2013).

The MK pool serves as an electron shuttle between cytoplasmic metabolism and membrane-bound electron transfer components, effectively linking intracellular respiration to periplasmic redox processes. Unlike many aerobic model organisms that use ubiquinone, G. sulfurreducens employs a menaquinone-based system more suited to anaerobic environments (Butler et al., 2006). Electrons from MKH₂ are transferred to inner membrane-bound multiheme cytochromes such as ImcH and CbcL, which are selectively active under different redox conditions (Meng et al., 2013). These cytochromes relay electrons to a periplasmic pool of small triheme c-type cytochromes, notably PpcABCDE (Coppi et al., 2007).

These periplasmic cytochromes diffuse freely within the periplasm and serve as electron shuttles between the inner membrane and outer membrane conduits. While this segment of the pathway does not contribute further to PMF, it plays a critical role in redox homeostasis. Without effective electron disposal, the MK pool becomes over-reduced, stalling NADH oxidation and halting energy generation at Complex I (Meng et al., 2013). Constraint-based models identified redundant, parallel pathways for acetate assimilation and electron transport away from the MK pool that sustain electron transfer when specific enzymes are disrupted (Segura et al., 2008). This redundancy, along with energy-conserving proton translocation, ensures robust intracellular metabolism coupled to extracellular Fe(III) reduction (Mahadevan R. et al., 2006). This architecture—centered on cytochrome-mediated transfer across the cell envelope—is a hallmark of G. sulfurreducens physiology and enables respiration of using a wide range of extracellular electron acceptors.

Geobacter sulfurreducens also possesses several alternative pathways to achieve redox balance. FrdCAB is a bifunctional fumarate reductase/succinate dehydrogenase that G. sulfurreducens can use to reduce fumarate intracellularly as a terminal electron acceptor (Butler et al., 2006; Frühauf-Wyllie and Holtmann, 2022). G. sulfurreducens further enhances its metabolic flexibility by utilizing alternative substrates such as formate and hydrogen as electron donors, dumping electrons into the redox pool via formate dehydrogenase and hydrogenases (Rotaru et al., 2012). These reactions supplement NADH and ferredoxin generation under specific growth conditions. Metabolic flux analyses show that intracellular fluxes dynamically adjust to maintain redox balance depending on the available electron donors and acceptors (Yang et al., 2010).

Overall, intracellular metabolism in G. sulfurreducens is optimized for electron production via acetate oxidation, with reducing equivalents entering the menaquinone-driven electron transport system (Figure 2). These steps link cytoplasmic metabolic pathways to electron transport pathways that end with the reduction of extracellular electron acceptors beyond the outer membrane. The presence of alternative metabolic pathways further enhances flexibility, allowing the organism to adapt to varying electron donor conditions.

The electron transport in the G. sulfurreducens periplasm occurs through a set of complementary, sometimes redundant, and dynamic cytochromes and their redox interaction. Within the inner membrane, several key cytochromes regulate the oxidation of menaquinol (MKH₂) and transfer electrons into the periplasm. The high-potential pathway involves the MHC ImcH, which oxidizes MKH₂, releases protons into the periplasm, and transfers electrons to periplasmic cytochromes such as PpcA with high affinity (Levar et al., 2017; Joshi et al., 2021; Pimenta et al., 2023). ImcH functions efficiently at redox potentials above −0.1 V and shares structural features with the NapC/NirT family, including a conserved quinone-binding site (Levar et al., 2014; Joshi et al., 2021). In contrast, the bc-type cytochrome complex CbcL facilitates MK oxidation under low redox potential conditions, supporting respiration when high-potential pathways such as ImcH are inactive (Zacharoff et al., 2016; Joshi et al., 2021). Another cytochrome, CbcBA, is induced when more easily reduced electron acceptors are absent and participates in electron transfer at potentials near −0.2 V. This enables G. sulfurreducens to sustain respiration when energy availability is extremely low—conditions where most organisms would not survive—highlighting its ability to adapt to thermodynamically constrained environments (Joshi et al., 2021).

Electrons transferred from inner membrane cytochromes enter the periplasm through interactions with periplasmic MHCs. PpcA, a triheme MHC, serves as the primary electron shuttle within the periplasm, receiving electrons from ImcH and CbcL and facilitating their transfer to outer membrane electron transport systems (Lloyd et al., 2003; Antunes et al., 2022; Choi et al., 2022; Pimenta et al., 2023). The periplasmic cytochrome family (PpcA-E) compensates for the loss of individual members through functional redundancy, allowing electron transfer to proceed even when specific pathways are disrupted. Despite initial hypotheses suggesting specificity among these cytochromes for particular electron acceptors, deletion mutant studies indicate that multiple Ppc cytochromes compensate for each other, ensuring robust electron transfer in diverse environments (Choi et al., 2022).

Biochemical studies of PpcA-E show that these periplasmic electron carriers transiently interact with each other, transferring charge and equilibrating redox potential, resulting in a redundant and dynamic periplasmic electron transfer pool (Ferreira et al., 2024). Elimination of all five cytochromes abolished soluble Fe(III) citrate reduction, confirming the necessity of periplasmic electron transfer for linking inner membrane electron flow to the outer membrane conduits. Expression of PpcA alone partially restored Fe(III) reduction in a dose-dependent manner using an inducible promoter, further supporting the redundancy of PpcA-E and the capacity to reconstitute function through single components (Ueki et al., 2017). Similarly strains retaining any one Ppc paralog (ABCDE) were able to reduce Fe(III) citrate, indicating that each of these individual cytochromes were sufficient to support EET under those conditions and suggesting a lack of strict specialization (Choi et al., 2022). In contrast, when grown on electrodes as the terminal electron acceptor, a mutant lacking the periplasmic MHCs, PpcA-E, and PgcA, an unrelated MHC that also shuttles electrons across the periplasm, exhibited wild type-like current production and similar electrode-associated biomass, suggesting that other, as yet unidentified, electrode-specific pathways exist for periplasmic electron transport (Choi et al., 2022).

Beyond electron transport, periplasmic cytochromes contribute to energy conservation and efficiency. PpcA has been shown to exhibit redox-Bohr effects, which regulate proton coupling during electron transfer, optimizing bioenergetic efficiency (Silva et al., 2021). Additionally, transient binding interactions between inner membrane and periplasmic cytochromes, including those observed between PpcA and ImcH, (Pimenta et al., 2023), or CbcL, (Antunes et al., 2022), suggest that transient electron transfer complexes allow electrons to be passed quickly and efficiently between proteins without needing strong, permanent binding.

Modular electron transfer pathways extend from the inner membrane to periplasmic cytochrome interactions, allowing G. sulfurreducens to fine-tune its respiratory processes in response to environmental redox conditions (Figure 2). Comparative proteomic studies have revealed that cytochrome expression shifts dynamically in response to different electron donors, such as acetate or formate, highlighting metabolic adaptability (Mollaei et al., 2021). This ability to balance multiple electron transfer pathways through the periplasm while optimizing energy conservation is a key feature of G. sulfurreducens metabolism, enabling survival in diverse anaerobic environments.

Recent advances have transformed our understanding of EET, particularly in G. sulfurreducens, where polymerized multiheme c-type cytochromes (MHCs) have been established as the primary conductive appendages mediating long-range electron transport. The discovery that OmcS, OmcE, and OmcZ form polymerized nanowires, and the resolution of their molecular structures, has provided a mechanistic foundation for interpreting electron transport from the outer membrane to insoluble electron acceptors. Yet, significant questions remain regarding the physiological roles, regulation, and assembly mechanisms of these cytochrome filaments.

Key scientific challenges include determining the conditions under which specific MHC nanowires are produced and function, identifying their membrane anchoring and electron entry/exit points, and resolving whether monomeric and polymeric forms play distinct roles. The mechanisms underlying cytochrome secretion and nanowire biogenesis—whether mediated by pilins, porin complexes, or proteolytic processing—remain incompletely understood. Moreover, it is unclear whether G. sulfurreducens secretes additional, as-yet-uncharacterized nanowires, or to what extent its secretion mechanisms are conserved across diverse microbial taxa. The complexity of regulatory and functional redundancy among cytochrome families also complicates efforts to assign discrete roles to individual components.

Technological challenges persist as well. There is a need for improved in situ methods to identify and characterize MHC nanowires under environmentally relevant conditions, as well as strategies to manipulate their properties for bioelectronic applications. High-resolution imaging, redox potential mapping, and single-cell expression tools could help disentangle the complex, condition-dependent roles of individual cytochromes. A particularly promising area is the heterologous expression of MHC nanowires in industrially relevant host organisms, such as E. coli or S. oneidensis. Successfully reconstituting nanowire assembly and electron transfer in such chassis could enable scalable, tunable, and autonomously assembled bioelectronic devices. However, this effort will require coordinated expression of cytochrome maturation factors, biogenesis systems, regulatory proteases, and secretion machinery proteins—many of which remain poorly defined even in G. sulfurreducens.

Future research should prioritize mechanistic dissection of nanowire secretion and anchoring, systematic mapping of EET network redundancy under fluctuating redox conditions, and development of synthetic biology tools to engineer and control MHC assembly and electron flow. Addressing these challenges will not only resolve fundamental questions in microbial respiration but also unlock new possibilities in bioenergy, bioremediation, and engineered living electronics.