Probing the Role of Metal Coordination and pH in Assembly and Function of Cytochrome Nanowires

Abstract

Geobacter sulfurreducens produces protein nanowires for extracellular electron transfer during respiration and interspecies electron exchange to survive in environments that lack soluble electron acceptors. It was previously thought that nanowires were Type IV pili comprised by PilA monomers, but we showed via electron cryo-microscopy (cryo-EM) that nanowires instead constitute a helical polymer of the six-heme outer membrane cytochrome OmcS, yielding a periodic arrangement of hemes over the micrometer scale of the filaments (Cell, 2019, 177, 361). This 3.7 Å-resolution structure revealed several features which appear to stabilize this polymer, including close stacking of covalently bound heme ligands within ∼ 3.5-6 Å of each other, and axial coordination of a heme metal center in each subunit by a histidine residue in the adjacent subunit. These OmcS filaments represent to our knowledge the first micrometer-scale cytochrome polymer naturally evolved for electron transfer, but the mechanism of this polymerization remains unknown. Furthermore, lowering the pH of nanowire samples enhances their conductivity and improves heme stacking, yet the structural changes in OmcS underlying this conformation change are unclear. Here, we develop a method to obtain OmcS nanowires and monomers in high purity. We then combine cryo-EM with spectroscopic methods such as UV-vis and circular dichroism to examine the changes in OmcS polymers and their inter-subunit interactions as a result of changes to pH, further enabling tuning of the conductivity of purified microbial nanowires. Furthermore, we explore the spectroscopic features and biochemical properties of OmcS monomers after reversing their polymerization, to obtain a mechanistic understanding of nanowire assembly that will aid engineering of synthetic protein nanowires for bioelectronics. This research was supported by Career Award from Burroughs Welcome Fund, NIH New Innovator award NSF CAREER award and DARPA (to N.S.M.).