In vivo Electrochemistry of C‐Type Cytochrome‐Mediated Electron‐Transfer with Chemical Marking

Abstract

In subsurface environments, certain types of microbes employ a unique metabolic strategy to respire on the naturally abundant iron-based minerals. Metal-reducing bacteria, such as those of the genus Shewanella and Geobactor, have a significant quantity of redox proteins located in their outer membrane (OM), and they transfer electrons from the cell surface to attached Fe oxides. 3] This is the terminal step in metabolism, and is an essential process to eliminate the excess electrons generated during the microbial oxidation of organic matter. Fe oxide respiration is considered to play an important role in the global biogeochemical cycling of iron. This process has also gained increasing attention for its potential application in mediatorless microbial fuel cells (MFCs). A great deal of research has been focused on measuring and proving bacterial extracellular electron-transfer (ET) respiration. The decaheme c-type cytochromes (c-Cyts), such as OmcA and MtrC, have been predicted to mediate ET to Fe oxides or electrodes. Up until this point, however, electrochemical and spectroscopic characterization of OM c-Cyts has been strictly limited to studies with purified proteins, although ET in living systems might be different from ET in purified proteins. In the whole-cell system, c-Cyts form a membrane-associated protein complex, and protein–protein and protein– membrane interactions would largely affect the energetics and kinetics of extracellular respiratory ET reactions. The main obstacle for the whole-cell study lies in the shortage of methods to differentiate c-Cyts from a number of uncharacterized biological molecules. Moreover, the extreme complexity and dynamic properties of living cells impede our ability to utilize knowledge developed in studies with purified proteins. Herein we report the control of the electronic states of OM c-Cyts in living cells of Shewanella by using an axial-coordination reaction on the heme groups of c-Cyts. We are the first to electrochemically identify OM c-Cyts and determine their ET kinetics under living conditions by using the specific binding affinity of nitric monoxide (NO). Our results reveal the existence of an respiratory ET chain with unusually high efficiency at the outer cell-membrane/electrode interfaces. Extracellular ET in living cells of Shewanella loihica PV-4 was monitored by using a single-chamber, three-electrode system with lactate as the carbon source and electron donor, as described in our previous studies. A tin-doped indium oxide electrode (ITO) was used as the working electrode and was placed on the bottom surface of the reactor. The cells were inoculated into the reactor under potentiostatic conditions at 0.4 V (versus the standard hydrogen electrode, SHE) for 25 h to prepare an electrode with attached cells. The optical microscopy image showed the formation of a thin layer (approximately 2 mm in length and 0.5 mm in width) that consisted of rod-shaped cells ; most of the cells attached horizontally to the electrode surface, not through the apex of their rod-like shape. Viability of the cells on the ITO electrode was confirmed by the generation of a microbial current (Figure S1 in the Supporting Information). As shown in the cyclic voltammograms (CVs, Figure 1), strain PV-4 exhibited one redox wave with a