Charge Regulation in a Rieske Proton Pump Pinpoints Zero, One, and Two Proton-Coupled Electron Transfer

The degree to which redox-driven proton pumps regulate net charge during electron transfer (ΔZ_ET) remains undetermined due to difficulties in measuring the net charge of solvated proteins. Values of ΔZ_ET can reflect reorganization energies or redox potentials associated with ET and can be used to distinguish ET from proton(s)-coupled electron transfer (PCET). Here, we synthesized protein "charge ladders" of a Rieske [2Fe-2S] subunit from Thermus thermophilus (truncTtRp) and made 120 electrostatic measurements of ΔZ_ET across pH. Across pH 5-10, truncTtRp is suspected of transitioning from ET to PCET, and then to two proton-coupled ET (2PCET). Upon reduction, we found that truncTtRp became more negative at pH 6.0 by one unit (ΔZ_ET = -1.01 ± 0.14), consistent with single ET; was isoelectric at pH 8.8 (ΔZ_ET = -0.01 ± 0.45), consistent with PCET; and became more positive at pH 10.6 (ΔZ_ET = +1.37 ± 0.60), consistent with 2PCET. These ΔZ_ET values are attributed to protonation of H154 and H134. Across pH, redox potentials of TtRp (measured previously) correlated with protonation energies of H154 and H134 and ΔZ_ET for truncTtRp, supporting a discrete proton pumping mechanism for Rieske proteins at the Fe-coordinating histidines.