Pivotal role of the strictly conserved aromatic residue F15 in the cytochrome c7 family

Joana M. Dantas, Leonor Morgado, Yuri Y. Londer, Ana P. Fernandes, Ricardo O. Louro, P. Raj Pokkuluri, Marianne Schiffer, Carlos A. Salgueiro

Research output: Contribution to journalArticle

11 Citations (Scopus)

Abstract

Cytochromes c7 are periplasmic triheme proteins that have been reported exclusively in d-proteobacteria. The structures of five triheme cytochromes identified in Geobacter sulfurreducens and one in Desulfuromonas acetoxidans have been determined. In addition to the hemes and axial histidines, a single aromatic residue is conserved in all these proteins-phenylalanine 15 (F15). PpcA is a member of the G. sulfurreducens cytochrome c7 family that performs electron/proton energy transduction in addition to electron transfer that leads to the reduction of extracellular electron acceptors. For the first time we probed the role of the F15 residue in the PpcA functional mechanism, by replacing this residue with the aliphatic leucine by site-directed mutagenesis. The analysis of NMR spectra of both oxidized and reduced forms showed that the heme core and the overall fold of the mutated protein were not affected. However, the analysis of 1H-15N heteronuclear single quantum coherence NMR spectra evidenced local rearrangements in the a-helix placed between hemes I and III that lead to structural readjustments in the orientation of heme axial ligands. The detailed thermodynamic characterization of F15L mutant revealed that the reduction potentials are more negative and the redox-Bohr effect is decreased. The redox potential of heme III is most affected. It is of interest that the mutation in F15, located between hemes I and III in PpcA, changes the characteristics of the two hemes differently. Altogether, these modifications disrupt the balance of the global network of cooperativities, preventing the F15L mutant protein from performing a concerted electron/proton transfer.

Original languageEnglish
Pages (from-to)11-24
Number of pages14
JournalJournal of Biological Inorganic Chemistry
Volume17
Issue number1
DOIs
Publication statusPublished - 1 Jan 2012

Keywords

  • Electron transfer
  • Geobacter
  • Multiheme cytochrome
  • NMR Site-directed mutagenesis

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