Date of Award

Fall 2022

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Molecular Biophysics and Biochemistry

First Advisor

Malvankar, Nikhil

Abstract

Proteins are commonly known to transfer electrons over distances limited to a few nanometers. However, many biological processes require electron transport over far longer distances. For example, some soil and sediment bacteria transport electrons over hundreds of micrometers to even centimeters. In particular, Geobacter sulfurreducens uses extracellular electron transfer (EET) to move respiratory electrons from the cytoplasm to external electron acceptors such as minerals or external electrodes in biofilm growth. These terminal acceptors are mere microns to as far as thousands of times the length of a cell away in biofilms. The exact mechanisms for the EET pathway and the long-range charge transport remain unclear. Cytochromes OmcS and OmcZ have been found to be of critical importance to G. sulfurreducens respiration over the years. It is known that OmcS is important for growth on minerals, and that OmcZ is critical for high current production during growth on electrodes. Recently, the discovery that both proteins form micrometer long nanowires in vivo has prompted an investigation into how each nanowire contributes to cellular metabolic processes, in contrast to previous studies focused on monomers. Understanding the electrochemical and electronic properties of microbial nanowires will help to elucidate their role in EET. Furthermore, light-induced microbial electron transfer has potential for efficient production of value-added chemicals, biofuels, and biodegradable materials owing to diversified metabolic pathways. Thus, both nanowires and biofilms should be investigated as biocompatible photoconductive materials for efficient electronic interface between microbes and electrodes. To achieve better understanding of long-range charge transport in proteins I used individual amyloid protein crystals with atomic-resolution structures as a model system. I perform contact-free measurements of intrinsic electronic conductivity using a 4-electrode approach. I find hole transport through micrometer-long stacked tyrosines at physiologically relevant potentials. Notably, the transport rate through tyrosines (105 s-1) is comparable to cytochromes. Combined experimental and computational studies reveal that proton-coupled electron transfer confers conductivity through energetics of the proton acceptor, a neighboring glutamine. To understand the role of both nanowires in EET by G. sulfurreducens I used electrochemical studies to remeasure their redox potentials as nanowires. I find through spectroelectrochemistry that the macroscopic midpoint potential of the OmcS nanowire is physiologically relevant at -0.130 V vs SHE, and it has a wide active redox range. The resulting redox landscape across a subunit of the nanowire follows an overall nearly thermoneutral pattern, consistent with the need to conserve energy for micrometer long electron transport. These results help to revise OmcS’s role in EET as a protein that can favorably accept electrons from the periplasm and transport them to minerals in the soils. I find that OmcZ nanowires have three redox peaks that range from -70 to -281 mV which indicates unique heme grouping in OmcZ. I investigate how differential expression of nanowires over time affects biofilm conductivity and redox potential in strains of wild type, ΔomcS, and ΔomcZ. I find that OmcZ abundance is correlated with an increase in metabolic current and conductance of biofilms. I find that electrochemical gating of wild type and ΔomcS matches the gating of OmcZ nanowires very well and find that OmcZ has greater than 400-fold gate effect compared to OmcS. Finally, I show that living biofilms of Geobacter sulfurreducens use nanowires of cytochrome OmcS as intrinsic photoconductors. Photoconductive atomic force microscopy shows >10-fold increase in photocurrent in purified individual nanowires. Photocurrents respond rapidly (<100 ms) to the excitation and persist reversibly for hours. Femtosecond transient absorption spectroscopy and quantum dynamics simulations reveal ultrafast (~200 fs) electron transfer between nanowire hemes upon photoexcitation, enhancing carrier density and mobility. Our work reveals a new class of natural photoconductors for whole-cell catalysis.

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