Abstract
Geobacter sulfurreducens bacterium exhibits an enormous respiratory versatility, including the utilization of several toxic and radioactive metals as electron acceptors. This versatility is also replicated in the capability of the most abundant cytochrome in G. sulfurreducens, the periplasmic triheme cytochrome PpcA, to reduce uranium, chromium and other metal ions. From all possible electron transfer pathways in G. sulfurreducens, those involved in the iron reduction are the best characterized to date. Previously, we provided structural evidence for the complex interface established between PpcA and the electron acceptor Fe(III)-citrate. However, genetic studies suggested that this acceptor is mainly reduced by outer membrane cytochomes. In the present work, we used UV-visible measurements to demonstrate that PpcA is able to directly reduce the electron acceptor ferric nitrilotriacetate (Fe-NTA), a more outer membrane permeable iron chelated form. In addition, the molecular interactions between PpcA and Fe-NTA were probed by Nuclear Magnetic Resonance (NMR) spectroscopy. The NMR spectra obtained for PpcA samples in the absence and presence of Fe-NTA showed that the interaction is reversible and encompasses a positively charged surface region located in the vicinity of the heme IV. Overall, the study elucidates the formation of an electron transfer complex between PpcA and a readily outer-membrane permeable iron chelated form. The structural and functional relationships obtained explain how a single cytochrome is designed to effectively interact with a wide range of G. sulfurreducens electron acceptors, a feature that can be explored for optimal bioelectrochemical applications.
Highlights
Geobacter species are highly abundant in natural subsurface environments where they can exchange electrons with insoluble acceptors, such as iron and manganese minerals (Reguera et al, 2005) or even with electrode surfaces (Holmes et al, 2004)
The conceptual model for the extracellular electron transfer in G. sulfurreducens considers that different proteins are involved in oxidation of the inner membrane quinols depending upon the redox potential of the final electron acceptor: the CbcLdependent pathway operates at or below redox potentials of −100 mV, whereas the ImcH-dependent pathway operates above this redox potential (Levar et al, 2014; Zacharoff et al, 2016)
To elucidate if one or more heme groups are directly involved in the reduction of ferric nitrilotriacetate (Fe-NTA), we studied the molecular interactions between the two molecules by Nuclear Magnetic Resonance (NMR)
Summary
Geobacter species are highly abundant in natural subsurface environments where they can exchange electrons with insoluble acceptors, such as iron and manganese minerals (Reguera et al, 2005) or even with electrode surfaces (Holmes et al, 2004). Whatever is the active respiratory pathway, the periplasmic cytochromes are always present and the cytochrome PpcA, which is the most abundant cytochrome in G. sulfurreducens (Ding et al, 2008; Morgado et al, 2010a) This cytochrome has 71 residues and contains three covalently bound low-spin heme groups with bis-histidinyl axial coordination, which are diamagnetic (S = 0) and paramagnetic (S = 1/2) in the reduced and oxidized forms, respectively (Morgado et al, 2010b; Dantas et al, 2011). Interacting interface regions between this cytochrome and electron acceptors were recently characterized, using a set of complementary biophysical techniques including stopped-flow kinetics, molecular docking, UV-visible and NMR measurements These studies allowed to identify the molecular complexes established between cytochrome PpcA and anthraquinone-2,6-disulfonate (AQDS), anthrahydroquinone2,6,-disulfonate (AH2QDS) and Fe(III)-citrate (Dantas et al, 2014a, 2015; Ferreira et al, 2017). Genetic and proteomic studies showed that the reduction of the humic substance analog (AQDS) and Fe(III)citrate by G. sulfurreducens and Shewanella oneidensis cells is essentially carried out by the outer membrane cytochromes (Gescher et al, 2008; Voordeckers et al, 2010; Bucking et al, 2012; Liu et al, 2015), suggesting that the direct reduction of these acceptors by PpcA is not significant
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