Molecular interaction studies revealed the bifunctional behavior of triheme cytochrome PpcA from Geobacter sulfurreducens toward the redox active analog of humic substances

Abstract

Humic substances (HS) constitute a significant fraction of natural organic matter in terrestrial and aquatic environments and can act as terminal electron acceptors in anaerobic microbial respiration. Geobacter sulfurreducens has a remarkable respiratory versatility and can utilize the HS analog anthraquinone-2,6-disulfonate (AQDS) as a terminal electron acceptor or its reduced form (AH2QDS) as an electron donor. Previous studies set the triheme cytochrome PpcA as a key component for HS respiration in G. sulfurreducens, but the process is far from fully understood. In this work, NMR chemical shift perturbation measurements were used to map the interaction region between PpcA and AH2QDS, and to measure their binding affinity. The results showed that the AH2QDS binds reversibly to the more solvent exposed edge of PpcA heme IV. The NMR and visible spectroscopies coupled to redox measurements were used to determine the thermodynamic parameters of the PpcA:quinol complex. The higher reduction potential of heme IV (-127mV) compared to that of AH2QDS (-184mV) explains why the electron transfer is more favorable in the case of reduction of the cytochrome by the quinol. The clear evidence obtained for the formation of an electron transfer complex between AH2QDS and PpcA, combined with the fact that the protein also formed a redox complex with AQDS, revealed for the first time the bifunctional behavior of PpcA toward an analog of the HS. Such behavior might confer selective advantage to G. sulfurreducens, which can utilize the HS in any redox state available in the environment for its metabolic needs.

Rational engineering of Geobacter sulfurreducens electron transfer components: a foundation for building improved Geobacter-based bioelectrochemical technologies

“…On the other hand, Voordeckers et al ( 2010 ) showed that the simultaneous deletion of genes coding for outer-membrane cytochromes OmcB, OmcS, OmcT, OmcE, and OmcZ yielded to a complete inhibition of AQDS reduction. Under the hypothesis that AQDS or humic substances are unable to access the periplasmic space of G. sulfurreducens , the interaction studies between AQDS and PpcA should be envisioned as working models (Dantas et al, 2014 , 2015a ). These models are even more relevant as no structural data are currently available for any of the outer membrane cytochromes mentioned above.…”

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

“…The structures are highly conserved suggesting that the interacting regions with putative redox partners can be mapped in experiments carried out with the protein in the reduced or oxidized state. This was indeed confirmed by NMR chemical shift perturbation studies between PpcA and the humic substance analog, which revealed that this molecule interacts in the same region with the cytochrome ( Dantas et al, 2014a , 2015 ).…”

“… 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.…”