Fuente: PubMed "microbial biotechnology" Appl Environ Microbiol. 2026 Jun 2:e0253625. doi: 10.1128/aem.02536-25. Online ahead of print.ABSTRACTThe freshwater, chemolithoautotrophic, Gram-negative bacterium Sideroxydans lithotrophicus ES-1 (ES-1) is thought to couple extracellular electron transfer (EET) to energy conservation through the Mto pathway and additional porin-cytochrome complexes, including Cyc2. The overall architecture and flexibility of ES-1 EET pathways remain poorly understood. Here, we investigated key components of the Mto pathway, focusing on the roles and interactions of the monoheme cytochromes MtoD and Slit_2494 (here designated as Periplasmic monoheme cytochrome A, PmcA), together with the inner-membrane tetraheme cytochrome ImoA. We performed the first biochemical and spectroscopic characterization of ImoA and PmcA, and investigated the interactions between multiple Mto pathway components using a combination of in vitro and in vivo approaches. We demonstrated that PmcA, but not MtoD, interacts with ImoA, and further show that PmcA, unlike MtoD, also interacts with MtoA. Our results identify PmcA as the physiological redox partner of ImoA, supported by favorable thermodynamics and direct protein-protein interactions. In contrast, the high redox potential of MtoD renders electron transfer to ImoA thermodynamically unfavorable. Instead, MtoD may transfer electrons to a high-potential downstream acceptor, such as a terminal oxidase. These findings refine and revise the previously proposed EET model for ES-1 and clarify the distinct roles of periplasmic cytochromes within the Mto pathway.IMPORTANCEFe(II)-oxidizing bacteria play an important role in the biogeochemical cycling of iron, representing a promising class of organisms for the development of novel biotechnological processes, including bioelectrosynthesis. These organisms perform extracellular electron transfer (EET) by acquiring electrons generated from the oxidation of Fe(II) outside the cell and transferring them into their internal metabolic processes. The understanding of EET pathways of these organisms is critical for harnessing and engineering their metabolic capabilities. In this study, we characterized biochemically and spectroscopically the cytochromes ImoA and PmcA from the Gram-negative Fe(II)-oxidizing bacterium Sideroxydans lithotrophicus ES-1 (ES-1). Through in vitro and in vivo interaction studies, we identified a direct interaction between ImoA and PmcA, but not with MtoD, which had previously been proposed as the physiological redox partner of ImoA within the Mto pathway. Furthermore, while MtoA did not interact with MtoD, it did show an interaction with PmcA, suggesting that PmcA may serve as an electron shuttle between outer and inner membrane components. These findings revise the current model of the Mto pathway and reveal a versatile electron transfer network in ES-1. This work provides critical insights into the electron transfer architecture of neutrophilic Fe(II)-oxidizing bacteria and broadens our understanding of EET strategies that can be leveraged for bioelectrosynthesis in microbial electrochemical technologies.PMID:42227757 | DOI:10.1128/aem.02536-25