In this work, the effect of pH on the functional mechanism of the triheme cytochromes PpcA and PpcD from Gs was evaluated. The thermodynamic parameters listed on for the fully reduced and protonated proteins, which include the heme reduction potentials, the pKa values of the redox-Bohr center, heme-heme redox interactions, and redox-Bohr interactions, were used to determine the heme oxidation profiles and the fractional contribution of the 16 microstates at different pH values. This allows to compare the individual heme oxidation profiles inside and outside the physiological pH range and to demonstrate a correlation between the functional properties of these cytochromes and the physiological pH range for cellular growth.
From the heme reduction potential values listed in , the order of oxidation of the heme groups can be established for the fully reduced and protonated protein, which is 1-3-4 for PpcA and 1-4-3 for PpcD. However, both proteins display important redox-Bohr interactions, being the largest one observed for heme 4. All the redox-Bohr interactions are negative, which is expected in electrostatic terms, since the removal of proton(s), upon deprotonation of the redox-Bohr center, lowers the affinity for electrons (lower reduction potential) by the heme groups. This is reflected in the heme reduction potentials of the fully reduced deprotonated proteins, which can be obtained by the simple sum of the heme reduction potentials of the fully reduced and protonated proteins with their respective redox-Bohr interactions. In the case of PpcA, the heme reduction potentials for the fully reduced and deprotonated protein are −186, −169, and −183
mV for hemes 1, 3, and 4, respectively. Comparison of these values with those obtained for the correspondent protonated form () clearly shows that the order of oxidation is different in both situations: 1-4-3 (deprotonated protein) and 1-3-4 (protonated protein). For PpcD, the same scenario is observed: the heme reduction potentials of the reduced and deprotonated protein are more negative (−184, −162, and −202
mV for hemes 1, 3, and 4, resp.), and the order of oxidation is affected by the deprotonation of the redox-Bohr center (4-1-3 versus
1-4-3). This analysis shows that the redox properties of PpcA and PpcD are modulated by the solution pH (redox-Bohr effect). However, the redox-Bohr effect is functionally relevant only if observed at physiological pH range for cellular growth.
To best of our knowledge, the optimal pH for Gs
growth has not yet been determined. Kim and Lee [21
] studied the effect of the initial pH on the growth rates of Gs
cultures utilizing fumarate as electron acceptor in the pH range of 5.5–6.8. Within this range, higher rates were observed at pH 6.8, which were reduced at pH 6.4 and completely inhibited at pH 5.5 [21
]. In addition to this non-electrode-respiring conditions, several electrode-respiring studies have shown a pH drop inside Gs
biofilms with a concomitant decrease in the measured current [22
]. Experiments carried out with anode-respiring bacteria with high presence of Gs
cells have shown that in the pH range 6–8, maximum current density was achieved at pH 8 and dropped continually to pH 6 [22
]. These studies also suggested that at pH < 6Gs
metabolism is inhibited.
In a previous work, we have used pH 7.5 as representative value for Gs
physiological pH [20
]. In the present work, we aimed to evaluate the significance of the redox-Bohr effect on the functional mechanism of PpcA and PpcD by studying the individual heme oxidation profiles and the fractional contribution of the microstates at a broader pH range. Thus, in order not to confuse literature, in the present work, the individual heme oxidation profiles and fractional contribution of microstates of both proteins were determined at pH 5.5 and 9.5 and compared with the previous analysis carried out at pH 7.5 (Figures and ). Then, a detailed analysis of the dominant PpcA and PpcD microstates was carried out in the pH range 5.5–9.5 ().
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 . The order of oxidation of the hemes is indicated (more ...)
Figure 3 Molar fraction of the 16 individual microstates (see ) 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 . Solid and (more ...)
Figure 4 Dependence of the molar fractions of PpcA and PpcD microstates with pH and solution potential (versus SHE) at 288K and 250mM ionic strength. The molar fractions of the individual microstates were determined using the parameters listed (more ...)
The heme oxidation profiles described in show that, at each pH value, the shape of the redox curves is substantially different from a pure Nernst curve and the several crossovers clearly indicate that the electron affinity of each heme is modulated by the heme-heme redox interactions, as protein oxidation progresses. The redox interactions are all positive, which is expected in electrostatic terms, and reflect the stabilization of the reduced state of one heme by the removal of one electron from a neighboring one. From the comparison of the individual heme oxidation profiles at different pH values, it is clear that the heme apparent midpoint reduction potentials eapp (i.e., the point at which the oxidized and reduced fractions of each heme group are equally populated) are different due to redox-Bohr interactions (). The shape of each heme oxidation curve is therefore a result of the interplay of both heme-heme and redox-Bohr interactions.
In the case of PpcA, at pH 5.5, the eapp values of hemes 3 and 4 are similar. However, at high pH, the deprotonation of the redox-Bohr center lowers considerable the eapp value of heme 4 (largest redox-Bohr interaction) bringing it closer to that of heme 1. The modulation of the individual heme oxidation profiles is also observed for PpcD, though yielding a distinct result. In fact, due to the similarity of the eapp values of hemes 1 and 4 at low pH, the deprotonation of the redox-Bohr center yields a more notorious separation of the three curves being heme 4 the one with smaller eapp value.
The effect of the protonation/deprotonation of the redox-Bohr center can be further rationalised from the fractional contribution of each microstate (). The fractional contribution of these microstates for PpcA and PpcD at pH 5.5, 7.5, and 9.5 provides functional mechanistic insights on the electron transfer pathways of the proteins (). The analysis of this figure shows that the relevant microstates are quite distinct at different pH values. In the case of PpcA, several microstates dominate the intermediate stages of oxidation either at pH 5.5 or pH 9.5. At pH 7.5, stage 0 is dominated by the protonated form P0H and stage 1 is dominated by the oxidation of heme 1 (P1H) while keeping the acid-base center protonated. Stage 2 is dominated by the oxidation of heme 4 and deprotonation of the acid-base center (P14), which remains deprotonated in stage 3 (P134). Therefore, at pH 7.5, a route is defined for the electrons within PpcA: P0H → P1H → P14 → P134, whereas at the other pH values there is no coherent path. Moreover, it is also clear that, associated with the favoured electron transfer pathway at physiologic pH, a deprotonation occurs as one electron is transferred between oxidation stages 1 and 2 () suggesting that microstates P14 and P1H are the physiological forms of PpcA. This mechanistic information, which can only be obtained from a detailed microscopic analysis shows how selected microstates could confer directionality of events: PpcA microstate P14 can uptake electrons and a weakly acidic proton from the donor associated with the cytoplasmic membrane, originating the microstate P1H. The pKa value of this proton (pKa 8) corresponds to the pKa value of oxidation stage 1 (). As discussed above, upon protonation of the redox-Bohr center, the reduction potential of the hemes becomes less negative. The loss of electron reducing power is used to lower the pKa of the proton so that when meeting the physiological downstream redox partner, microstate P1H donates less reducing electrons and a more acidic proton (pKa 7.2, which corresponds to the pKa value of oxidation stage 2—see ). This is now sufficiently acidic to be released in the periplasm, and microstate P14 is now ready to initiate a new energy transduction cycle.
PpcD can also couple the transfer of electrons and protons at physiologic pH, though by a different pathway (). The oxidation stage 0 is dominated by the protonated form P0H
. However, the microstates of oxidation stage 1 are overcomed by the P0H
curve, which intercepts first the P14
curve. This microstate (P14
) dominates the oxidation stage 2, whereas P134
dominates stage 3. Thus, for this cytochrome, a different preferential route for electrons is established, favouring a proton-coupled 2
transfer step between oxidation stages 0 and 2: P0H
. As for PpcA, at pH 5.5 and 9.5, no coupling between electron and proton transfer is observable for PpcD ().
Several studies using biofilms on electrodes have shown that electron transfer out of Gs
cells is detectable above potentials of about −200
standard hydrogen electrode [22
]. Thus, in the present work, we further determined the microstates of PpcA and PpcD showing the largest molar fraction values in the redox potential window −200
mV to −30
mV and also in the pH range 5.5–9.5 (). This analysis shows that PpcA and PpcD are not fully reduced nor fully oxidized and thus are functionally active (i.e., capable of receiving and donating electrons) in that redox potential and pH range. Additionally, it is interesting to note that the microstates pairs that can couple e−
transfer are essentially detectable at the pH range for which most studies with Geobacter
cells have been carried out. In particular, PpcA, which is highly abundant in Gs
, being most likely the reservoir of electrons destined for outer surface [28
], can perform e−
energy transduction in the pH range 6.5 to 8.5 albeit at different redox potential values (). It should be emphasized that the data presented in was obtained for purified proteins and that the transposition of these data to Gs
cells grown in cultures or biofilms should take into account that in these more complex systems, other variables such as the presence of the other cellular components, metabolic status, and environmental conditions (e.g., ionic strength) can alter slightly the working range of these proteins. Studies on Gs
biofilms on electrodes showed that cells have a Nernstian response around −150
]. Since PpcA is functionally active at this redox potential (), it can be hypothesized that this response is mainly PpcA driven. Under this hypothesis and since PpcA is also able to perform e−
energy transduction at pH values around 7, a possible functional mechanism would involve the transfer of one electron and one proton from the quinone oxidoreductase being the second electron transfer back to the quinone pool.
Overall, the work presented here shows that PpcA and PpcD display the adequate functional properties to perform e−
energy transduction but only within the pH range 6.5–8.5. This might represent additional mechanisms contributing to the H+
electrochemical potential gradient across the periplasmic membrane that drives ATP synthesis and might also explain why Gs
cells become metabolically inactive at pH 6 or lower [22