Determinants of Multiheme Cytochrome Extracellular Electron Transfer Uncovered by Systematic Peptide Insertion

The
multiheme cytochrome MtrA enables microbial respiration by
transferring electrons across the outer membrane to extracellular
electron acceptors. While structural studies have identified residues
that mediate the binding of MtrA to hemes and to other cytochromes
that facilitate extracellular electron transfer (EET), the relative
importance of these interactions for EET is not known. To better understand
EET, we evaluated how insertion of an octapeptide across all MtrA
backbone locations affects Shewanella oneidensis MR-1
respiration on Fe­(III). The EET efficiency was found to be inversely
correlated with the proximity of the insertion to the heme prosthetic
groups. Mutants with decreased EET efficiencies also arose from insertions
in a subset of the regions that make residue–residue contacts
with the porin MtrB, while all sites contacting the extracellular
cytochrome MtrC presented high peptide insertion tolerance. MtrA variants
having peptide insertions within the CXXCH motifs that coordinate
heme cofactors retained some ability to support respiration on Fe­(III),
although these variants presented significantly decreased EET efficiencies.
Furthermore, the fitness of cells expressing different MtrA variants
under Fe­(III) respiration conditions correlated with anode reduction.
The peptide insertion profile, which represents the first comprehensive
sequence–structure–function map for a multiheme cytochrome,
implicates MtrA as a strategic protein engineering target for the
regulation of EET.