Molecular interactions between Geobacter sulfurreducens triheme cytochromes and the electron acceptor Fe(iii) citrate studied by NMR

Abstract

Proteomic and genetic studies have identified a family of five triheme cytochromes (PpcA-E) that are essential in the iron respiratory pathways of Geobacter sulfurreducens. These include the reduction of Fe(iii) soluble chelated forms or Fe(iii) oxides, which can be used as terminal acceptors by G. sulfurreducens. The relevance of these cytochromes in the respiratory pathways of soluble or insoluble forms of iron is quite distinct. In fact, while PpcD had a higher abundance in the Fe(iii) oxides supplanted G. sulfurreducens cultures, PpcA, PpcB and PpcE were important in Fe(iii) citrate supplanted cultures. Based on these observations we probed the molecular interactions between these cytochromes and Fe(iii) citrate by NMR spectroscopy. NMR spectra were recorded for natural abundance and N-enriched PpcA, PpcB or PpcE samples at increasing amounts of Fe(iii) citrate. The addition of this molecule caused pronounced perturbations on the line width of the protein's NMR signals, which were used to map the interaction region between each cytochrome and the Fe(iii) citrate molecule. The perturbations on the NMR signals corresponding to the backbone NH and heme methyl substituents showed that complex interfaces consist of a well-defined patch, which surrounds the more solvent-exposed heme IV methyl groups in each cytochrome. Overall, this study provides for the first time a clear illustration of the formation of an electron transfer complex between Fe(iii) citrate and G. sulfurreducens triheme cytochromes, shown to be crucial in this respiratory pathway.

Biomolecular Interaction Studies Between Cytochrome PpcA From Geobacter sulfurreducens and the Electron Acceptor Ferric Nitrilotriacetate (Fe-NTA)

“… Comparison of the most affected residues in the redox complexes established between PpcA from G. sulfurreducens and the elctron acceptors Fe-NTA (this work), Fe-citrate ( Ferreira et al, 2017 ) and AQ(H 2 )DS ( Dantas et al, 2014a , 2015 ). The residues highlighted in red and blue correspond to those that showed a decrease in the peak data height above 40% or a significant deviation in their chemical shift, respectively.…”

“…Interestingly, previous studies have also shown that the heme IV region in the cytochrome PpcA from G. sulfurreducens was also involved in the formation of redox complexes with other electrons acceptors, namely the humic substance analog (AQDS) and Fe-citrate ( Dantas et al, 2014a , 2015 ; Ferreira et al, 2017 ). A comparison of the interacting polypeptide regions between PpcA and the three electron acceptors are summarized in Figure 7 .…”

“…Interacting interface regions between this cytochrome and electron acceptors were recently characterized, using a set of complementary biophysical techniques including stopped-flow kinetics, molecular docking, UV-visible and NMR measurements. These studies allowed to identify the molecular complexes established between cytochrome PpcA and anthraquinone-2,6-disulfonate (AQDS), anthrahydroquinone-2,6,-disulfonate (AH 2 QDS) and Fe(III)-citrate ( Dantas et al, 2014a , 2015 ; Ferreira et al, 2017 ). The results obtained also suggested that the cytochrome PpcA may function as terminal reductase for AQDS and Fe(III)-citrate fractional populations that are able to transverse the outer membrane and reach the periplasm.…”

Electron Paramagnetic Resonance Characterization of the Triheme Cytochrome from Geobacter sulfurreducens

“…The heme midpoint potentials are found to lie within a rather narrow range, from the lowest of Heme I equal to −162 mV to the highest potential of Heme IV, around −133 mV. 26 Nuclear magnetic resonance (NMR) chemical shift perturbation measurements have demonstrated the presence of specific binding sites on the PpcA surface for small molecule redox substrates 27,28 and partner redox proteins. 29 Similarly, PpcA dimerization induced by anionic porphyrin binding also points toward an electrostatic surface that is mapped for recognition at a single surface location.…”

“…Evidence for site-selective tuning of the heme cofactors in PpcA is seen by the heme methyl group proton NMR chemical shift patterns that are distinct, and readily distinguishable for each of the three heme cofactors. 27,28 The differing chemical shift patterns observed for the heme cofactors in both the cytochromes c 3 and c 7 families are significant since they provide a measure of the modulation of electron spin densities at atomic sites on the porphyrin ring which are found to be characteristically tuned for each heme cofactor. 31−35 A direct approach for characterization of site-specific electronic structure configuration of ferric hemes is through electron paramagnetic resonance (EPR) spectroscopy.…”