Development of a Novel Platform for Spectroelectrochemical Investigation of Geobacter Cytochromes Involved in Uranium Reduction
Authors: Awate Bhushan , Worden Robert Mark , Reguera Gemma 
Published: February 2020

Metal-reducing subsurface bacteria like Geobacter sulfurreducens can reductively precipitate water-soluble uranium salts. However, very little information is available about this bacteria's uranium reduction mechanism. Most of the metabolic investigations are based on genetic analysis, where specific proteins are either deleted or overexpressed to elucidate its role in the electron transfer process. However these approaches to identify the electron carriers are limited because mutation of one gene can cause multiple phenotypic changes resulting in alterations of the native uranium reductase machinery. Yet, these limitations can be bypassed through the use of nanoscale in vitro platforms that mimic the Geobacter cell envelope electron carrier machinery. Therefore, we constructed nanostructured biomimetic interfaces equipped with Geobacter’s most abundant and conserved electron carrying cytochromes (periplasmic cytochrome PpcA and outer membrane cytochrome OmcB) to mimic the cell membrane electron transfer mechanism and adapted it to the platform capable of spectroelectrochemical determination. Spectroelectrochemical techniques combines electrochemistry and spectroscopic measurements, and offers advantage offering capability to assess changes in specific molecules in the system as a function of electric potential. We built working electrode form a transparent slide coated with gold (~100 Ao thickness) and covered with self assembled monolayer (SAM) of thiolipids.  Formation of SAM on the gold electrodes shielded the protein solution from degradation at the electrode surface, and furthermore provided us with the capability of controlling the access of species in solution towards working electrode. Redox activity of Geobacter cytochrome c PpcA was measured electrochemically by technique like cyclic voltammetry and chronoamperometry, where changes in current at the working electrode are monitored as a function of applied potential. High transparency of the working electrode allowed us to measure reduction of PpcA spectrophotometrically by presence of three characteristic absorption bands, called Soret or gamma peak which shift from ~408 in oxidized form to ~420 in reduced form, alpha (α) peak at ~ 552 nm and beta (β) at ~ 521 nm. The cytochrome was immobilized onto a mixed self assembled monolayer of alkanethiols on a gold electrode. The ratio of carboxy acid terminating alkane group to alcohol terminating alkane group was optimized for the immobilization of protein in active conformation. Immobilized PpcA was characterized electrochemically by cyclic voltammetry, chronoamperometry and square wave voltammetry. Immobilized PpcA showed stable electrochemical response and was capable of being redox active in the immobilized state. The midpoint redox potential of immobilized PpcA was -372 mV vs. Ag/AgCl, also within the orders obtained for PpcA in solution (-374 mV vs. Ag/AgCl). Thus the immobilized PpcA at the SAM electrode was capable of direct electron transfer without any need of mediator resulting precise control over electrochemical potential.

Based on the hypothesis that PpcA may be transfer electrons produced in respiration to OmcB as part of Geobacter’s electron transport chain, we attempted to demonstrate electron transfer from PpcA to OmcB in optically transparent thin layer cell. Addition of OmcB-containing membrane vesicles to the PpcA, yielded a small increase in the CV peak of PpcA. Since OmcB membrane vesicles, are not capable of direct electron transfer to and from the electrode, the increase in the current can be attributed to the catalytic reduction of OmcB by reduced PpcA. Redox spectra of the cytochromes monitored in the multipotential step experiments showed additional evidence of reduction of OmcB from PpcA. Change were observed in the Soret Peak at 410 and 420 nm, and 552 peaks as a function of electrode potential.

The biomimetic system assembled here was intended to simulate electron transfer within Geobacter’s cell envelope, in which electrons produced in respiration are transferred from the inner membrane (mimicked by the SAM coated gold electrode) through the periplasm (mimicked by the PpcA layer bound to the SAM) to outer membrane cytochromes (mimicked by the OmcB-containing microsomes. This study’s establishment of a biomimetic redox-active cell envelope (BRACE) provides a new research platform with which to study Geobacter electron transport to extracellular electron acceptors. This nanoplatform integrating the key components of Geobacter’s electron transfer machinery will allow us to study the activity of native redox components in a controllable system. The information obtained about the redox interaction between cytochrome provides interesting insights about the mechanism of electron transfer in Geobacter which may lead to more successful strategies for groundwater uranium fixation.