Characterization of Axial and Proximal Histidine Mutations of the Decaheme Cytochrome MtrA from Shewanella sp. Strain ANA-3 and Implications for the Electron Transport System

Abstract

c Extracellular respiration of solid-phase electron acceptors in some microorganisms requires a complex chain of multiheme ctype cytochromes that span the inner and outer membranes. In Shewanella species, MtrA, an ϳ35-kDa periplasmic decaheme c-type cytochrome, is an essential component for extracellular respiration of iron(III). The exact mechanism of electron transport has not yet been resolved, but the arrangement of the polypeptide chain may have a strong influence on the capability of the MtrA cytochrome to transport electrons. The iron hemes of MtrA are bound to its polypeptide chain via proximal (CXXCH) and distal histidine residues. In this study, we show the effects of mutating histidine residues of MtrA to arginine on protein expression and extracellular respiration using Shewanella sp. strain ANA-3 as a model organism. Individual mutations to six out of nine proximal histidines in CXXCH of MtrA led to decreased protein expression. However, distal histidine mutations resulted in various degrees of protein expression. In addition, the effects of histidine mutations on extracellular respiration were tested using ferrihydrite and current production in microbial fuel cells. These results show that proximal histidine mutants were unable to reduce ferrihydrite. Mutations to the distal histidine residues resulted in various degrees of ferrihydrite reduction. These findings indicate that mutations to the proximal histidine residues affect MtrA expression, leading to loss of extracellular respiration ability. In contrast, mutations to the distal histidine residues are less detrimental to protein expression, and extracellular respiration can proceed.

Mechanistic Insights into Dye-Decolorizing Peroxidase Revealed by Solvent Isotope and Viscosity Effects

“…This is not surprising, as mutation of the axial ligand in heme proteins (H215 in El DyP) has been known to result in the failure of heme incorporation. 33–34 SDS-PAGE (Figure S1-A in SI) reveals that the molecular weight (MW) of El DyP is 35 kDa, which is consistent with the calculated value. Since the presence of the His 6 tag did not interfere with enzyme activity, it was not removed prior to further biochemical studies.…”

Multi-heme cytochromes provide a pathway for survival in energy-limited environments

“…As verified in some iron-reducing bacterial strains, the identified multi-heme cytochromes may form an integral protein complex across the OM in D. ferrophilus IS5. Because the cytochromes encoded by DFE_449 and DFE_461 detected in the extracted OM fraction were predicted to be at the periplasm; and these genes are located in close genomic proximity to DFE_450 and DFE_464 , which are estimated to encode the extracellular cytochromes (table S4); DFE_449 and DFE_461 may encode OM-bound periplasmic cytochromes that perform a similar functional role to the S. oneidensis MtrA, a periplasmic decaheme cytochrome in the OM MtrCAB cytochrome complex that is important for metal reduction in Shewanella species ( 23 , 24 ). Transcriptome analysis also revealed comparable expression levels for the genes DFE_448 to DFE_451 and DFE_461 to DFE_465 ( Fig.…”

Evidence for Horizontal and Vertical Transmission of Mtr-Mediated Extracellular Electron Transfer among the Bacteria

“…The MtrCAB outer membrane conduit has been genetically and physiologically implicated in EET among Shewanella oneidensis , Shewanella sp. ANA-3, Aeromonas hydrophila , and Vibrio natriegens ( 28 , 31 , 33 , 34 , 45 , 46 ). Accordingly, to begin investigating the prevalence of these genes among other bacteria, we searched for homologs of MtrCAB across the entire domain.…”