Revealing the structural origin of the redox-Bohr effect: the first solution structure of a cytochrome from Geobacter sulfurreducens

Abstract

Gs (Geobacter sulfurreducens) can transfer electrons to the exterior of its cells, a property that makes it a preferential candidate for the development of biotechnological applications. Its genome encodes over 100 cytochromes and, despite their abundance and key functional roles, to date there is no structural information for these proteins in solution. The trihaem cytochrome PpcA might have a crucial role in the conversion of electronic energy into protonmotive force, a fundamental step for ATP synthesis in the presence of extracellular electron acceptors. In the present study, 15N-labelled PpcA was produced and NMR spectroscopy was used to determine its solution structure in the fully reduced state, its backbone dynamics and the pH-dependent conformational changes. The structure obtained is well defined, with an average pairwise rmsd (root mean square deviation) of 0.25 Å (1 Å=0.1 nm) for the backbone atoms and 0.99 Å for all heavy atoms, and constitutes the first solution structure of a Gs cytochrome. The redox-Bohr centre responsible for controlling the electron/proton transfer was identified, as well as the putative interacting regions between PpcA and its redox partners. The solution structure of PpcA will constitute the foundation for studies aimed at mapping out in detail these interacting regions.

Proteins involved in electron transfer to Fe(III) and Mn(IV) oxides by Geobacter sulfurreducens and Geobacter uraniireducens

“…sulfurreducens that folds into a crescent-shaped nanowire of 12 haems (Morgado et al, 2012;Pokkuluri et al, 2011).…”

Solution structure of a mutant of the triheme cytochrome PpcA from Geobacter sulfurreducens sheds light on the role of the conserved aromatic residue F15

“…1). The distribution of the number of restraints is not uniform along the protein sequence, as heme groups attached to positions 30, 54 and 68 show many long-distance contacts, a feature in common with the wild-type protein [21].…”

“…With the exception of the first two residues, the NMR signals of all the other backbone, side chains and heme substituents were assigned following the same methodology used for the wild-type protein [21]. To avoid overlap with polypeptide amide protons, the heme signals were assigned in the NMR spectra acquired for PpcAF15L samples prepared in 2 H 2 O, according to the strategy described by Turner and co-workers [33].…”

“…The functional characterization of PpcA showed that the protein has the necessary thermodynamic properties to convert electronic energy into proton motive force, a fundamental step that might contribute to the proton electrochemical gradient across the bacterial cytoplasmic membrane [20]. The mechanism that allows PpcA to perform such concerted e − /H + transfer is driven by the protonation/deprotonation of a redox-Bohr center, which was assigned to the heme IV propionate 13 (P 13 IV , according to the IUPAC nomenclature) [21]. The protonation/deprotonation of this center leads to conformational changes in residues located in its vicinity, which were mapped on the solution structure of PpcA [21].…”

“…The mechanism that allows PpcA to perform such concerted e − /H + transfer is driven by the protonation/deprotonation of a redox-Bohr center, which was assigned to the heme IV propionate 13 (P 13 IV , according to the IUPAC nomenclature) [21]. The protonation/deprotonation of this center leads to conformational changes in residues located in its vicinity, which were mapped on the solution structure of PpcA [21]. Given the significance of the residue Phe 15 , it was replaced by site-directed mutagenesis with a leucine in the triheme cytochrome PpcA (hereafter designated PpcAF15L) and the impact of the substitution on the redox properties of the protein was evaluated [19].…”