ABSTRACT
The degree to which redox-driven proton pumps regulate net charge during electron transfer (ΔZET) remains undetermined due to difficulties in measuring the net charge of solvated proteins. Values of ΔZET 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 ΔZET 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 (ΔZET = -1.01 ± 0.14), consistent with single ET; was isoelectric at pH 8.8 (ΔZET = -0.01 ± 0.45), consistent with PCET; and became more positive at pH 10.6 (ΔZET = +1.37 ± 0.60), consistent with 2PCET. These ΔZET 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 ΔZET for truncTtRp, supporting a discrete proton pumping mechanism for Rieske proteins at the Fe-coordinating histidines.
