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

Abstract

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.

Keywords: Shewanella oneidensis MR-1; dissociation constant; electron transfer; electrostatics; extracellular respiration; paramagnetic NMR; periplasmic cytochromes.

FIGURE 1

¹H-1D NMR spectral changes of the signal from methyl 18¹ belonging to heme IV of small tetraheme cytochrome (STC) in the presence of increasing amounts of octaheme tetrathionate reductase (OTR), illustrating the data used in the chemical shift perturbation analysis. The samples were prepared in 20 mM phosphate buffer pH 7.6, with 100 mM KCl, at 25°C. The methyl group is labeled using the IUPAC-IUB nomenclature for hemes. The Roman numeral corresponds to the order of heme binding to the polypeptide chain. The R values correspond to the molar ratios of [OTR]/[STC].

FIGURE 2

Binding curves of periplasmic cytochromes from SOMR-1 that show interactions monitored by ¹H-1D-NMR spectra: OTR and STC (A) and DmsE and STC (B). The chemical shift perturbations of the heme methyl signals are plotted as a function of the molar ratio of the interacting proteins. Solid triangles and solid squares represent the 7¹ methyl and 18¹ methyl of heme IV of STC, respectively; open triangles represent the 7¹ methyl of heme II of STC. The solid lines represent the best global fit to the 1:1 binding model [equation (1)].

FIGURE 3

Competition binding curves between STC and FccA with MtrA monitored by ¹H-1D NMR spectra. The chemical shift perturbations of the heme methyl signals are plotted as a function of the molar ratio of the interacting proteins. Filled symbols represent the experiment performed in the absence of FccA (Fonseca et al., 2013), whereas open symbols represent the experiment in the presence of 360 μM of FccA to ensure that more than 90% of MtrA is bound to FccA. The solid lines represent the best global fit to the 1:1 binding model [equation (1)] as reported by Fonseca et al. (2013).

FIGURE 4

UV–visible spectroscopy of reduced periplasmic cytochromes in the presence of excess fumarate and catalytic amounts of FccA illustrating the absence of electron transfer between OTR with FccA. UV-visible spectrum of the cytochrome as purified (a), after reduction with sodium dithionite (b) and subsequent addition of fumarate (c). After addition of FccA to the mixture, spectra were acquired at 0 min (d), 2.5 min (e), and 5 min (f). The samples were prepared in 20 mM phosphate buffer pH 7.6, with 100 mM KCl, at 25°C.

FIGURE 5

Electrostatic potential mapping on the protein’s surface of (A) OTR (PDB code 1S3P) and (B) ccNiR (PDB code3UBR) from SOMR-1. Electrostatic potentials were calculated considering a fully oxidized state for these cytochromes. The Roman numerals correspond to the order of heme binding motifs in the polypeptide chain.

FIGURE 6

Interactions between the most abundant periplasmic cytochromes (STC and FccA), and outer membrane complex MtrDEF (A), periplasmic nitrite reductases OTR and ccNiR (B) and outer membrane complex DmsEFABGH (C). The arrows in bold indicate the interactions that occur between the cytochromes and point to the possible docking site. Black arrows indicate data from this work and gray arrows indicate data previously reported (Fonseca et al., 2013). The dissociation constants (Kd) corresponding to each interaction are indicated next to their respective arrow. Interactions that were not detected experimentally are represented by a crossed-out arrow. The Roman numerals correspond to the order of heme attachment to the polypeptide chain. Cytochrome representations were made with PyMOL using the structures of STC (PDB code 1M1Q), FccA (PDB code 1D4D), OTR (PDB code 1S3P), and ccNiR (PDB code 3UBR). For CymA the model was made with SWISS-MODEL (Arnold et al., 2006; Kiefer et al., 2009) using as template the structure of NrfH (PDB code 2J7A).