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

Abstract

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.

Observation of super-Nernstian proton-coupled electron transfer and elucidation of nature of charge carriers in a multiredox conjugated polymer

“…From the measured Pourbaix diagram of each of the three redox processes in BBL-P, we have discovered that the PCET response is super-Nernstian, exhibiting two protons per one electron transferred. Although super-Nernstian PCET has previously been observed in some hydrated transition metal oxides 9–12 and metalloproteins, 4,13,14 our present results represent the first observation of super-Nernstian PCET behavior in a purely organic material/polymer.…”

“…4,8 Very few exceptions from the stoichiometric proton/ electron ratios, or deviation from Nernstian behavior, have been observed. 4,[9][10][11][12][13][14] In particular, hydrated inorganic oxides with redox-active transition metal centers (e.g. nickel-borate, iridium, ruthenium) can afford a super-Nernstian shi (70-120 mV pH −1 ), [9][10][11][12] whereby the additional proton uptake can be attributed to the presence of water molecules since thermal annealing of such oxides to rigorously remove excess moisture resulted in a conventional Nernstian shi.…”

“…nickel-borate, iridium, ruthenium) can afford a super-Nernstian shi (70-120 mV pH −1 ), [9][10][11][12] whereby the additional proton uptake can be attributed to the presence of water molecules since thermal annealing of such oxides to rigorously remove excess moisture resulted in a conventional Nernstian shi. 10 In addition to inorganic oxides, biological molecules such as ironcoordinating histidine based metalloprotein have also produced super-Nernstian shi 4,13,14 due to its unique molecular structure which features separate electron sinks (i.e. ferrous/ ferric redox centers) and proton sinks (i.e.…”