Fine Tuning of Redox Networks on Multiheme Cytochromes from Geobacter sulfurreducens Drives Physiological Electron/Proton Energy Transduction

Abstract

The bacterium Geobacter sulfurreducens (Gs) can grow in the presence of extracellular terminal acceptors, a property that is currently explored to harvest electricity from aquatic sediments and waste organic matter into microbial fuel cells. A family composed of five triheme cytochromes (PpcA-E) was identified in Gs. These cytochromes play a crucial role by bridging the electron transfer from oxidation of cytoplasmic donors to the cell exterior and assisting the reduction of extracellular terminal acceptors. The detailed thermodynamic characterization of such proteins showed that PpcA and PpcD have an important redox-Bohr effect that might implicate these proteins in the e(-)/H(+) coupling mechanisms to sustain cellular growth. The physiological relevance of the redox-Bohr effect in these proteins was studied by determining the fractional contribution of each individual redox-microstate at different pH values. For both proteins, oxidation progresses from a particular protonated microstate to a particular deprotonated one, over specific pH ranges. The preferred e(-)/H(+) transfer pathway established by the selected microstates indicates that both proteins are functionally designed to couple e(-)/H(+) transfer at the physiological pH range for cellular growth.

Figure 1

Electronic distribution scheme for a triheme cytochrome with a proton-linked equilibrium showing the 16 possible microstates. PpcA and PpcD are structurally similar to tetraheme cytochromes c ₃, with the exception that heme 2 and the corresponding fragment of the polypeptide chain are absent. Thus, to be consistent with the literature, the heme groups are numbered 1, 3, and 4 according to the order of attachment to the CXXCH motif in the polypeptide chain. The blue and red circles correspond to the protonated and deprotonated microstates, respectively. Hexagons represent heme groups, which can be either reduced (black) or oxidized (white). The microstates are grouped, according to the number of oxidized hemes, in four oxidation stages connected by three one-electron redox steps. P _0H and P ₀ represent the reduced protonated and deprotonated microstates, respectively. P _ijkH and P _ijk indicate, respectively, the protonated and deprotonated microstates, where i, j, and k represent the heme(s) that are oxidized in that particular microstate.

Figure 2

Heme oxidation fractions for PpcA (a) and PpcD (b) at different pH values. The curves were calculated as a function of the solution reduction potential (versus SHE) using the parameters listed in Table 1. The order of oxidation of the hemes is indicated by the arrow.

Figure 3

Molar fraction of the 16 individual microstates (see Figure 1) of PpcA (a) and PpcD (b) at different pH values. The curves were calculated as a function of the solution reduction potential (versus SHE) using the parameters listed in Table 1. Solid and dashed lines indicate the protonated and deprotonated microstates, respectively. For clarity, only the relevant microstates are labeled.

Figure 4

Dependence of the molar fractions of PpcA and PpcD microstates with pH and solution potential (versus SHE) at 288 K and 250 mM ionic strength. The molar fractions of the individual microstates were determined using the parameters listed in Table 1, and those showing the largest molar fraction are represented. The regions where proteins can perform e⁻/H⁺ transfer and e⁻ transfer are highlighted in dark gray and gray, respectively. Regions where the protein is fully reduced or fully oxidized are indicated in light gray.