
==== Front
J Am Chem Soc
J Am Chem Soc
ja
jacsat
Journal of the American Chemical Society
0002-7863
1520-5126
American Chemical Society

37486967
10.1021/jacs.3c03006
Article
Charge Regulation in a Rieske Proton Pump Pinpoints Zero, One, and Two Proton-Coupled Electron Transfer
https://orcid.org/0000-0003-2021-5298
Koone Jordan C. †
Simmang Mikaela ‡
Saenger Devin L. ‡
Hunsicker-Wang Laura M. *‡
https://orcid.org/0000-0001-8265-5833
Shaw Bryan F. *†
† Department of Chemistry and Biochemistry, Baylor University, Waco, Texas 76706, United States
‡ Department of Chemistry, Trinity University, San Antonio, Texas 78212, United States
* lhunsick@trinity.edu
* bryan_shaw@baylor.edu
24 07 2023
02 08 2023
24 07 2024
145 30 1648816497
22 03 2023
© 2023 The Authors. Published by American Chemical Society
2023
The Authors
https://creativecommons.org/licenses/by-nc-nd/4.0/ Permits non-commercial access and re-use, provided that author attribution and integrity are maintained; but does not permit creation of adaptations or other derivative works (https://creativecommons.org/licenses/by-nc-nd/4.0/).

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.

Division of Chemistry 10.13039/100000165 CHE 2203441 Welch Foundation 10.13039/100000928 W-0031 Welch Foundation 10.13039/100000928 AA-1854 document-id-old-9ja3c03006
document-id-new-14ja3c03006
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pmcIntroduction

The chemical and physical mechanisms by which metalloproteins tune rates and free energies of electron transfer (ET)1,2 or proton-coupled electron transfer (PCET)3−8 are not fully understood. In recent years, we have used capillary electrophoresis (CE) and “protein charge ladders” to make the first measurements of charge regulation during single ET and PCET in metalloproteins.9,10 Charge regulation (or charge compensation) is the ability of a system to resist changes in net charge (Z) upon accepting, donating or binding to any charged species (proton, electron, metal, ligand, or protein).11−14 Charge regulation during ET in metalloproteins (denoted ΔZET) occurs from shifts in the pKa of ionizable residues or tightly bound solvent or cofactors in response to (or during) changes in metal oxidation state.9,11,15

To date, values of ΔZET have been measured for four metalloproteins: cytochrome c (Cyt c), azurin (Az), superoxide dismutase-1 (SOD1), and myoglobin (Mb).9,10 Values of ΔZET are protein-specific, and so far, measured values vary between ∼0 and ∼ –1 upon reduction with a single electron. For example, we previously found that ΔZET = −1.19 ± 0.02 for Cyt c, whereas ΔZET = −0.05 ± 0.08 for SOD1.9,10 The SOD1 enzyme regulates net charge perfectly upon reduction due to redox-driven protonation of its histidine bridge.10 Cytochrome c exhibits low charge regulation likely reflecting a functional need for low reorganization energy.9 Values for Az and Mb are in between: AzΔZET = −0.51 ± 0.02 and MbΔZET = −0.62 ± 0.06.9 Charge regulation in Az and Mb were attributed to increases in the pKa of multiple noncoordinating residues upon reduction (3 residues in Az and 11 in Mb).

Positive values of ΔZET (upon reduction) have not yet been reported. We expect such values to exist for redox processes in which the transfer of one electron is coupled to the transfer of two protons (2PCET). Two-proton coupled electron transfer processes have not been reported in metalloproteins, per se. They have been described theoretically by Hammes-Schiffer in organic amidinium-carboxylate interfaces and have been observed electrochemically in reactions between dihydroxynaphthalenes and reactive oxygen species.17,18

The current study measured ΔZET in a truncated form of the Rieske protein from Thermus thermophilus (denoted truncTtRp).19 The truncTtRp protein lacks 8 N-terminal and 9 C-terminal residues. A majority of the 17 truncated residues are hydrophobic and nonionizable (except for two E, one Y, and two R). In this paper, the net charges of oxidized and reduced truncTtRp (Zox, Zred) are measured over a pH range of 5.50–10.60 (Thermus thermophilus can survive from pH ∼ 5 to 9.6).20

Rieske proteins are proton pumps essential to almost all living organisms, as they are components of the electron transfer chain in mitochondria, bacteria, cyanobacteria, chloroplasts, and algae.21,22 Rieske proteins have a conserved [2Fe–2S] cluster, in which the redox active Fe is coordinated by two histidine residues (H134 and H154 in truncTtRp). The redox-inactive Fe is coordinated by two cysteine residues.23,24 Pioneering studies of truncTtRp suggest that H154 and H134 are sequentially deprotonated at increasing pH (to imidazolate) in Fe(III) but become reprotonated upon reduction to Fe(II).19,25 The pKa of these two histidine residues have been approximated with NMR,2,26,27 Raman spectroscopy,28−31 UV–vis and CD spectroscopy,32,33 along with cyclic voltammetry:25,34 pKa ≈ 7.40–7.87 for Fe(III)-H154 and pKa ≈ 9.10–9.84 for Fe(III)-H134. The pKa of both histidines are ≈12.30–12.50 in the Fe(II) state. These pKa values suggest that the truncTtRp protein engages in predominately single ET at pH ≤ ∼7.40–7.85 and PCET at pH ≈ 7.40–9.84, where His154 becomes protonated upon reduction. At pH ≈ 9.84–12.5, it is hypothesized that two protons are coupled to electron transfer (i.e., H154 and H134 are both protonated upon reduction). We refer to this latter process as “2PCET.”

According to classical Marcus theory for electron transfer, the inner sphere and outer sphere reorganization processes that contribute to total reorganization energy (λ) occur prior to the electron jump.5,16 We, of course, do not know when proton transfer is occurring in the Rieske protein, relative to electron transfer. Moreover, in the case of 2PCET, we certainly do not know whether each of the two protons transfers at the same rate (presumably not). If the transfer of one or more protons happens before or during ET, then protonation should contribute to λ. If protonation occurs after electron transfer (e.g., in a slower process, possibly involving a conformational change), then protonation should affect the redox potential (equilibrium free energy, ΔG°). Regardless of when protonation occurs relative to ET, it will likely affect the rate of ET, according to Marcus theory because the rate of ET depends upon both λ and ΔG°. Thus, the magnitude of ΔZET will likely affect the rate of ET by affecting λ and/or ΔG°.

Despite all that is known about Rieske proteins, it remains unclear how tightly regulated their redox processes are electrostatically and how the net protonation changes upon ET. For example, do residues outside the active site of truncTtRp adjust their pKa during single ET—like with azurin—but not during PCET, like with SOD1? That is, does proton coupling function as an electrostatic shield during ET to “hide” changes in the metal oxidation state from ionizable residues outside the active site? These questions can be directly answered by measuring ΔZET across pH. Quantifying ΔZET will also provide a stoichiometric picture of the net protonation of truncTtRp during redox cycling, which is required for understanding the mechanisms of proton pumping. In addition, truncTtRp provides a unique model system for quantifying ΔZET in a metalloprotein that appears to be capable of both single ET and PCET at the same metal site, albeit at different values of pH.

Results and Discussion

Formation and Characterization of Protein Charge Ladders

Measuring the net charge (Z) of a folded, solvated protein (at pH ≠ pI) is analytically challenging.11,12,35 Only a fraction of proteins (<40 by our estimate)36 have had their values of net charge directly measured in the folded, solvated state at pH ≠ pI. Isoelectric points represent only one value of Z (i.e., Z= 0) at one pH (i.e., pH = pI) and do not express the magnitude of charge at pH ≠ pI.11,35 In this study, “protein charge ladders” of truncTtRp were synthesized and analyzed with capillary zone electrophoresis (CE) to measure changes in the net charge of truncTtRp upon reduction of Fe(III) to Fe(II).9−11,36−38

A “protein charge ladder” is an electrostatic array of proteins with systematically altered surface charge, but similar shape.11 A protein charge ladder of any protein can be synthesized by acetylation with acetic anhydride, which neutralizes surface lysine-ε-NH3+ (Figure 1). Each acetylation alters mass by ∼42 Da and net charge by slightly less than −1.0 (e.g., ΔZAc ≈ 0.9) per modification due to charge regulation (Figure 1).39 When analyzed with CE, a protein charge ladder resolves into a self-calibrating, internally consistent electrophoretic array. Protein charge ladders and CE can quantify small changes in charge associated with ET. Here, each “rung” of the charge ladder represents a protein with a different number of acetylated lysines (Ac(N)) and, therefore, a different net surface charge. Plotting the linear correlation between the electrophoretic mobility of each rung against each Ac(N) produces an x-intercept that represents the net charge (Z) of the unacetylated protein divided by the change in charge per acetylation (Figure 1d).9−11,36 Importantly, these measurements are made in <10 min, using nanoliters of sample in freshly deoxygenated running buffer. Conceptually, this linear plot (i.e., the x-intercept) illustrates the number of theoretical R-NH3+ groups that must be added to this negatively charged protein to achieve a net charge of zero and a mobility of zero.

Figure 1 What is a protein “charge ladder” and how can it be used to measure changes in net charge (Z) associated with reduction of truncTtRp? (a) Formation of a protein charge ladder by acetylation of lysine with excess acetic anhydride. (b) The charge ladder is first characterized with mass spectrometry to determine the number of acetylations (Ac(N)). (c) Capillary electrophoresis is then used to analyze the electrophoretic mobility (μ) of the oxidized (black) and reduced (red) protein charge ladders. (d) Finally, the linear plot of electrophoretic mobility (μ) of each rung versus Ac(N) is extrapolated to the x-intercept which equals: ZAc(0)/ΔZAc. Insert: UV–vis spectra of oxidized (black) and reduced (red) truncTtRp following addition of ∼100 mol equiv of sodium dithionite. Note, dithionite is instantly separated from the protein during electrophoresis in deoxygenated running buffer.

We first characterized protein charge ladders of truncTtRp with mass spectrometry (MS) to assess sites of acetylation and effects on structure prior to analysis with CE. Liquid chromatography (LC)-MS/MS of a tryptic-digested protein charge ladder of truncTtRp identified acetylation on over 80% of lysine residues without modification of other residues (i.e., His, Cys, Figure S1). Therefore, even though the charge ladder consists of ∼5 “rungs” (acetylated lysine), each rung consists of a mixture of lysine-acetyl regioisomers. Acetylation of 0 to 5 lysine in truncTtRp did not significantly disrupt the hydrophobic core of the protein, as illustrated by the similar rate of amide hydrogen–deuterium exchange (HDX) of each rung in 90% D2O (Figure 2, Table S1).

Figure 2 Amide H/D exchange of a truncTtRp protein charge ladder with zero to five acetylations. (a) Electrospray ionization mass spectra used to manually deconvolute the molecular weights at each time point. The +15-charge state, showing each acetylation, is inset. (b) Number of unexchanged amide hydrogens after 60 min in 90% D2O, as calculated from the difference in mass of native protein in D2O (after incubation for 60 min) and the mass of the perdeuterated (heat-denatured) protein. Here, error bars represent the standard deviation of mass values from three technical replicates.

Values of Z are determined from a protein charge ladder by plotting the mobility (μ) of each “rung” versus the number of acetylation(s) (Ac(N)) for that rung. At low rungs, a linear trend is observed (Figure 3). The x-intercept of this linear function equals the Z of the unmodified protein divided by the change in charge of each acetylation, ΔZAc (Figure 3). The net electrostatic charges of the oxidized FeIII-truncTtRp (Zox) and reduced FeII-truncTtRp (Zred) were measured across a pH range of 5.5–10.6 (caveats associated with measuring Z at pH > 9.5 are discussed below). Electropherograms collected at three critical pH values (pH 6.0, 8.8, and 10.6) are presented first (Figures 3–5), to showcase electrostatic effects at pH where either ET, PCET, or 2PCET appears to be singularly dominant, without significant contributions from the other two processes. Electropherograms at seven other pH values were also collected (Figure S2) and analyzed (Figure S3).

Figure 3 The net charge (Z) of truncTtRp protein is reduced by exactly one unit of charge upon single-electron transfer (ET) at pH 6.0. (a) Six replicate electropherograms with lysine-acetyl protein charge ladders of oxidized (black) and reduced (red) truncTtRp at pH 6.0 (20 mM MES). (b) Electrophoretic mobility (μ) of each “rung” versus the number of acetylated lysine, Ac(N). The x-intercept corresponds to the quotient of ZAc(0)/ΔZAc (i.e., the net charge of the unacetylated protein divided by the change in charge of each acetylation). Error bars represent the standard deviation of average mobility from replicate electropherograms. (c) Proposed ET reaction in truncTtRp when pH < pKa of H134 and H154.

Electron Transfer at pH 5.5–pH 7.0

At pH 6.0, truncTtRp is expected to engage in single electron transfer (uncoupled to proton transfer), according to previously determined pKa values of both H154 and H134. At pH 6.0, the net charge of FeIII-truncTtRp was measured to be Zox = −5.45 ± 0.02 (Figure 3a–b). Reduction of FeIII-truncTtRp to FeII-truncTtRp with sodium dithionite was confirmed via the disappearance of the Fe d–d transition band at 458 nm with UV–vis (Figure 1d). The total net charge of FeII-truncTtRp was measured to be Zred = −6.46 ± 0.14. The change in net charge following reduction of Fe(III) to Fe(II) was ΔZET = −1.01 ± 0.14 (Figure 3b). This ΔZET is consistent with an active site where both histidine residues are protonated in the Fe(III) and Fe(II) states (Figure 3c). Furthermore, this ΔZET demonstrates that truncTtRp does not regulate its net charge during redox cycling at pH 6.0, as do other metalloproteins (e.g., Az and Mb). Rather, truncTtRp behaves like Cyt c, with little charge regulation. Small deviations in ΔZET are seen throughout this pH range (i.e., pH 5.5–7.0) with values moving closer to 0.5 as the pKa of His154 is approached (Figures S2, S3).

Proton Coupled Electron Transfer at pH 7.4–pH 8.8

At pH 8.8, protonation of Fe-bound H154 upon reduction (pKa = 7.85)34 is expected to offset changes in charge associated with ET. At pH 8.8, the net charge of FeIII-truncTtRp was measured to be Zox = −9.73 ± 0.45 and that of FeII-truncTtRp was measured to be Zred = −9.74 ± 0.07 (Figure 4a–b). The overall change in net charge following reduction of Fe(III) to Fe(II) at pH 8.8 was ΔZET = −0.01 ± 0.45 (Figure 4b). This change in Z is consistent with the proposed active site at this pH (and with PCET), where one histidine residue is deprotonated to form an imidazolate in the oxidized Fe(III) state and reprotonated following reduction to Fe(II) (Figure 4c). This near-zero ΔZET suggests but does not prove that no other net changes in the pKa of ionizable residues occur in the protein upon reduction (that are large enough to alter net charge). Throughout the pH range of 7.4–8.2, values of ΔZET ranged from −0.87 ± 0.25 to −0.14 ± 0.15 (Figures S2, S3). This range of ΔZET is consistent with a mixture of both ET and PCET occurring upon reduction, where His154 can be in either a protonated (i.e., imidazole) or a deprotonated (i.e., imidazolate) state in the Fe(III) form.

Figure 4 Stoichiometric proton-coupled electron transfer (PCET) in truncTtRp at pH 8.8: oxidized and reduced proteins are isoelectric. (a) Six replicate capillary electropherograms with protein charge ladders of oxidized (black) and reduced (red) truncTtRp at pH 8.8 (20 mM MES). (b) Linear plot of electrophoretic mobility (μ) of each rung versus the number of acetylated lysine, Ac(N). The x-intercept corresponds to the quotient ZAc(0)/ΔZAc. Error bars represent the standard deviation of each replicate electropherogram. (c) Proposed PCET reaction when pH > pKa of H154.

Two-Proton Coupled Electron Transfer at pH 10.1 and 10.6

The net electrostatic charge of both the oxidized and reduced proteins was finally measured at 10.6, which is above the previously suggested pKa values of both His residues (Figure 5). Here, the Z of the FeIII-truncTtRp was measured to be Zox = −18.08 ± 0.50 and that of FeII-truncTtRp was measured to be Zred = −16.71 ± 0.33 (Figure 5b). Therefore, the truncTtRp protein becomes more net positive (less negative) upon the reduction of Fe(III) to Fe(II). The overall change in net charge following reduction of Fe(III) to Fe(II) at pH 10.6 was ΔZET = +1.37 ± 0.60 (Figure 5b). This ΔZET is consistent with an active site where both histidine residues are doubly deprotonated (i.e., two imidazolate) in the Fe(III) state, with two protons transferred to H154,134 upon electron transfer (“2PCET”, Figure 5c). At pH 10.1, ΔZET = 0.33 ± 0.22. This intermediate value suggests that there is a mixture of both PCET and 2PCET occurring, with H134 in protonated or deprotonated states in the Fe(III) state (Figures S2, S3). Previous experiments utilizing cyclic voltammetry suggest that if we were to continually increase the pH (i.e., pH > 12) we would see this process return to single ET (i.e., ΔZET = −1), where both histidines remain in the imidazolate forms in oxidized or reduced states.25,34

Figure 5 The truncTtRp protein becomes more positive upon reduction at pH 10.6: evidence of two protons coupled to electron transfer (2PCET). (a) Six replicate capillary electropherograms with protein charge ladders of oxidized (black) and reduced (red) truncTtRp at pH 10.6 (20 mM MES). (b) Plot of electrophoretic mobility (μ) of each “rung” versus the number of acetylated lysine, Ac(N). The x-intercept corresponds to the quotient of ZAc(0)/ΔZAc. Error bars represent the standard deviation of each replicate electropherogram. (c) A proposed 2PCET reaction of truncTtRp when pH > pKa of H154 and H134.

We now discuss a few caveats associated with our measurements at pH > 9.5. Note that the separation of each rung in the charge ladders gradually decreases as the pH increases from 5.5 to 10.6 (Figure S2). For example, the change in mobility (Δμ) imparted by each acetylation decreases by ∼0.2 per pH unit (Figure S4). This type of decrease in Δμ of a protein charge ladder as the protein becomes more net negatively charged can be explained by Debye–Hückel theory, as previously described by Whitesides and co-workers.11

Likewise, estimating the net charge of proteins at high pH (>10) becomes challenging because (i) each rung becomes less resolved and (ii) the change in charge per acetylation (ΔZAc) will eventually decrease from 0.90 as the pKa of lysine is reached. We do not know the pKa of lysine residues in this protein, but a survey of 155 folded proteins suggests that the lysine pKa varies from 6.50 to 12.12, with an average of 10.70.40 In this study, we used a constant value of ΔZAc = 0.90 to determine the net charge. If this value is much smaller than 0.90, at pH 10.6, the effects would not change the conclusions of the measurements. For example, if we used a ΔZAc of ∼0.50 to calculate Z at pH 10.6 (the highest pH used in this study), the ΔZET would still be positive. The two possible values of ΔZET are not statistically different (P = 0.059), i.e., ΔZET = +1.37 ± 0.60 (using ΔZAc = 0.90) and ΔZET = +0.77 ± 0.34 (using ΔZAc = 0.50). This result would still suggest that 2PCET is occurring at pH 10.6, regardless of the two possible values of ΔZAc used to calculate Zox/red.

Nevertheless, we conclude that the use of ΔZAc = 0.90 is appropriate as it yields the expected net charge of truncTtRp based on Henderson–Hasselbalch calculations of Zred using average pKa values for all ionizable residues (Figure 6a).40 In the reduced state, both Fe-coordinated His residues are always protonated below pH 12.5. The charge of truncTtRp is Zred = −9.28 ± 0.28 when a ΔZAc value of 0.50 is used (compared to a value of Zred = −16.71 ± 0.33 when ΔZAc = 0.90 is used). The theoretical (formal) value of Zred for truncTtRp at pH 10.6 is −14.7. Therefore, this theoretical Zred and our measured Zred are in better agreement when we use ΔZAc = 0.90 at all pH values.

Figure 6 Trends in Z and ΔZET over pH 5.5–10.6. (a) Values of Z vs pH for oxidized (black) and reduced (red) truncTtRp. The solid line represents theoretical Z of reduced truncTtRp calculated from the Henderson–Hasselbalch equation and experimentally determined pKa values for all ionizable residues. (b) Values of ΔZET vs pH for truncTtRp. The solid gray line represents the theoretical ΔZET upon reduction determined from literature pKa values for the two Fe-bound histidine residues (H154, H134). Error bars represent the standard deviation of charge values from 12 replicate electropherograms.

Biological Implications of ET, PCET, and 2PCET

Plotting the net charge of both the oxidized and reduced truncTtRp vs pH (and ΔZET vs pH) provides a bird’s eye view of the transitioning between ET, PCET, and 2PCET across pH (Figure 6a–b). The values of ΔZET vs pH appear to follow the Henderson–Hasselbalch equation with input from just the pKa values of H154 (7.87) and H134 (9.84) that were determined for truncTtRp by Hunsicker-Wang et al. (Figure 6b).32

Our results suggest that, at pH values of 5.5 and 6, truncTtRp conducts single ET without net proton transfer coupled at any residue in the protein. At this pH, truncTtRp cannot facilitate proton pumping per se. Moreover, there appears to be no contribution to ΔZET other than that imparted by the electron. That is, the truncTtRp protein behaves like Cyt c (and unlike Az, Mb, or SOD1) in this pH range, with no apparent net change in the pKa of ionizable residues upon ET that would be large enough to alter Z. This is notable, as there are 36 ionizable residues in the subunit with 2 residues within 12 Å of the redox center (and Fe-bound histidines) and 13 residues within 20 Å (Figure S5a). It is, of course, possible that the pKa of some residues increased and others decreased such that no net change in protonation (charge) can be observed. However, we favor an interpretation where the protonation states of no other ionizable residues change upon reduction.

In contrast, at pH 8.8, there is isoelectric (stoichiometric) PCET, presumably with little net adjustment in pKa of any other residues besides the Fe-bound histidine, which would produce a measurable change in net charge. Here, the electrostatic effect of Fe reduction appears to be perfectly offset by the protonation of His154. In this regard, the truncTtRp protein engages in redox behavior similar to that of the SOD1 protein—perfect charge regulation via protonation of a single metal-coordinating imidazolate. Thus, while these two proteins—SOD1 and truncTtRp are different in form, function, and chemistry—they are quite similar bioelectrically. These proteins are the only two proteins that we know that contain a His-imidazolate at physiological pH. In vivo, the protonation of H154 in FeII-truncTtRp would presumably come from quinol and be pumped across the cellular membrane to the periplasmic space (or inter membrane space in eukaryotes) upon oxidation to Fe(III).

Closer to physiological pH, e.g., pH 7.4 to 7.8, the ΔZET of truncTtRp ranges from −0.87 ± 0.25 to −0.47 ± 0.13 respectively (Figures S3, S4). These values suggest that the H154 residue is not fully deprotonated prior to reduction, as expected from the pKa. This suggests that there is not a stoichiometric acceptance of one proton per electron in this truncated protein. Rather, there is ∼0.2–0.5 stoichiometric equivalents of protons accepted by H154 per electron in truncTtRp (with H2O as a proton donor). This stoichiometry might be different in vivo, when TtRp is in complex. For example, previous studies have suggested that a conserved lysine residue in cytochrome b interacts (electrostatically) with both Fe-bound histidine residues in the Rieske protein when accepting an electron and proton from quinol.27,41,42 The close proximity of the positively charged lysine (∼6–10 Å from the Rieske center) could stabilize the imidazolate form of H154 and H134. This lowering of the pKa of H154 and H134 could possibly promote PCET and 2PCET processes and diminish uncoupled, single ET.

At slightly alkaline pH values (pH > 9.0), the truncTtRp protein can acquire two net protons for every electron accepted (i.e., a 2PCET process). These two net protons, presumably residing on His154 and His134, come from solvent in these in vitro experiments. Perhaps both would be derived from the same quinol in vivo or from two successively colliding quinols. In the quinol (Q) cycle of biological electron transport proton pumping is thought to occur via PCET processes involving Rieske proteins.43−45 Using a simple approximation based on the Henderson–Hasselbalch equation, we estimate that 0.001% to 36.5% of the truncTtRp protein is engaging in 2PCET from pH 5.10–9.60, the range at which Thermus thermophilus cells can grow.20 This 2PCET process is of course intriguingly similar to the 2:1 proton/electron stoichiometry of the Qo-site, where truncTtRp is found.46−48 However, our calculations pertain only to this isolated, truncated protein out of complex with its other binding partners. Therefore, we do not wish to speculate on the degree to which 2PCET is happening at Rieske centers in vivo.

Diffuse versus Discrete Charge Regulation

When considering that ionizable groups exist in a nonlinear cooperative network, it must be remembered that the Debye length over which charges sense one another (∼10 Å when ionic strength = 0.1) is expected to be longer inside the hydrophobic interior of proteins.11 If distal residues in the truncTtRp protein framework respond to reduction of Fe(III) by regulating charge, that is, if residues outside the active site adjust pKa upward (as we see with single ET proteins such as Az), then this could alter the pKa of H154. This alteration could result in the redox-driven protonation being spread out over multiple ionizable residues (“diffuse” charge regulation). Therefore, it might be a requirement of truncTtRp (and perhaps some other PCET proteins such as SOD1) that they do not regulate charge diffusely—that the pKa of metal-distal residues does not change—and that only the protonation of one critical residue changes during redox cycling (a metal-coordinating histidine in the case of SOD1 and truncTtRp). We of course do not know how general this model would be to other PCET proteins. We hypothesize that truncTtRp is able to discretely accept protons at H154 and H134, without diffuse protonation at other off-target ionizable residues, because there are no other ionizable residues near the active site (Figure S5a). This would suggest that substitution with ionizable residues near the active site might diminish discrete protonation (proton coupled electron transfer) and interfere with the ability of truncTtRp to pump protons.

The importance of ΔZET in the redox energetics of Rieske proteins

These simple measurements of charge can provide insights into the role of electrostatics in electron transfer proteins that engage in single ET or PCET. As first pointed out by Rees nearly 40 yeas ago, the role of a protein’s net charge correlates with its redox potential (E).49 Here, the formal (unmeasured) net charge of ∼40 proteins correlates with E by 23 mV per unit of charge.49 Positively charged proteins generally have more positive E than negatively charged proteins (with the midpoint at Z = ∼ –5).49

The current study suggests that ΔZET might be a critical factor in determining the E of redox proteins. The mechanisms by which proteins tune redox potentials of metal ions are not perfectly understood, but involve multiple chemical and physical factors inside and outside of the active site (e.g., active site geometry; metal type; and protein net charge).49,50 In the case of truncTtRp, there is a clear negative sigmoidal trend in E vs ΔZET, and a positive sigmoidal trend in E vs ZOx (Figure 7). Here, potentials were extracted from previously published graphs of E vs pH for the nontruncated TtRp25,34 (using WebPlotDigitizer to extract potentials at every 0.05 pH unit from pH 2.5–14). The sharp drop in E of TtRp coincided with the transition in truncTtRp from pure ET (i.e., ΔZET = −1) to PCET (ΔZET = 0) and 2PCET (ΔZET = +1). This drop in E is indicative of strong proton coupling to electron transfer in truncTtRp.5 Note that the E values are for full length TtRp and not truncTtRp. While the magnitude of E may be different for these proteins, we do not expect the pH profile to be different.

Figure 7 Relationship between charge regulation (ΔZET), protonation free energy of H154, H134 (ΔΔGH+) in truncTtRp, and redox potential (E) ofTtRp. (a) E vs SHE of TtRp vs ZOx (black circles) and ΔZET (white triangles) of truncTtRp across pH 5.5–10.6. The redox potentials of TtRp were extracted from a previous study.34 (b) E vs SHE of TtRp (black circles) and ΔΔGH+ (white circles) across pH 2.6–14. Red dashed lines indicate pKa of H154 and H134.

To explore any links between ΔZET and E we determined whether the pH dependence of E mirrored the combined protonation energies of H154 and H134. Here, the free energy required for the adjustments of both H154 and H134 upon single ET (denoted ΔΔGH+) was calculated from pH 5.5 to 11 (at increments of 0.1 pH units) using the equation: ΔG = 2.3RT(pH – pKa) as previously described.9,51 For these calculations, the absolute value of the difference between the pH of solvent and the pKa are calculated as we are only interested in the energy required to change the protonation state and not necessarily the direction of protonation (i.e., deprotonation or protonation). This linearized function is why the plot of ΔΔGH+ vs pH appears faceted or segmented (Figure 7b).

Somewhat to our surprise, the values of E for TtRp and ΔΔGH+ for truncTtRp are nearly superimposable across pH (Figure 7b). Therefore, it is reasonable to surmise that if E mirrors ΔΔGH+ across pH, and ΔΔGH+ drives ΔZET across pH, then E and ΔZET are also linked across pH. Further, E and ΔZET vs pH show similar trends (Figure S5b) where the sigmoidal fit of ΔZET and E fall to zero, coincidentally, at the same pH value (the pKa of H134). This coincidence is of course dependent upon the electrode used when measuring redox potentials (i.e., a value of 0 mV for SHE is −244 mV for SCE and −280 mV for NCE). In conclusion, these data suggest that E in TtRp is regulated by the two Fe-bound histidines, with no net (detectable) changes in the pKa of other ionizable residues upon ET.

As expected, there is a sigmoidal trend between ΔZET and Z across pH for truncTtRp (Figure 8a). We considered these electrostatic properties of truncTtRp in the context of our previous measurements of Z and ΔZET for Az, bovine SOD1, human SOD1, Mb, and Cyt c.9,10 For all of the various proteins and redox states that we have measured—including truncTtRp in each regime (ET, PCET, and 2PCET)—there is a gross correlation between ΔZET and ZOx with obvious outliers (Figure 8b). In general, the trend suggests that proteins with high negative charge are more likely to engage in some type of proton-coupled ET than proteins with net positive charges. The truncTtRp protein demonstrates this point across 20 units of charge and five units of pH (Figure 8a).

Figure 8 Correlation between net electrostatic charge (ZOX) of several metalloproteins and charge regulation following electron transfer (ΔZET). (a) Plot of ΔZET vs ZOx for truncTtRp across pH 5.5–10.6 (20 mM MES) fit with a sigmoidal function. (b) Plot of ΔZET vs ZOx of several metalloproteins previously measured, including truncTtRp from this study. The pH of solvent for each measurement is in parentheses. Error bars represent the standard deviation from each replicate electropherogram.

Regarding PCET processes in proteins, it is tempting to postulate that the proton being transferred might function, in general, as an electrostatic shield to protect other distal ionizable residues from sensing the new electrostatic environment at the reduced metal ion. After all, this shielding would seemingly prevent distal, noncoordinating residues from shifting pKa (which would otherwise increase outersphere reorganization energy). This study suggests that this reasoning is not necessarily correct if the protein does not contain any distal ionizable residues to begin with. For example, the truncTtRp does not regulate charge during reduction with a single electron at pH 6.0, because charge is decreased by almost exactly one unit (ΔZET = −1.01 ± 0.14). This suggests that no residues are changing pKa in response to ET. At pH 8.8, the change in net charge is almost exactly zero (ΔZET = −0.01 ± 0.45). If this proton was acting as an electrostatic shield for other distal ionizable residues, a ΔZET other than −1 would be seen at pH 6.0. The only residue whose pKa seems to be adjusted, based on decades of work, is His154. A future test of this hypothesis—that PCET proteins are tuned to not regulate charge at distal sites—could involve the measurement of ΔZET in a binuclear metalloprotein capable of doing single ET at one metal and PCET at the other metal site, at the same pH.

Materials and Methods

Recombinant Expression and Purification of the Truncated Rieske Protein from Thermus thermophilus (truncTtRp)

Recombinant truncTtRp was expressed in E. coli and purified as previously described.19E. coli was grown in TB containing 100 μg/mL ampicillin to an OD600 of ∼1.0 AU. The cells were then induced with 0.4 mM IPTG, supplemented with 150 μM cysteine and 150 μg/mL ferric ammonium citrate, shaken for 18–20 h, and then pelleted via centrifugation at 5000 × g for 5 min at 4 °C. This pellet was resuspended in 25 mL of lysis buffer per liter of TB. The lysis buffer contained 10 mM Tris-HCl, pH 8.0, 4 mg/mL lysozyme, 40 units/mL DNase, 3 units/mL RNase, 2 mM phenylmethylsulfonylfluoride, and 0.1% Triton X-100. Following resuspension with lysis buffer, cells were incubated for 60 min at 30 °C. The solution was then centrifuged at 12000 g for 15 min at 4 °C and the pellet discarded. The solution was dialyzed against 25 mM Tris-HCl, pH 8.0. The protein was purified with consecutive anion exchange chromatography (DE52 resin, HiTrap Q HP) and size exclusion chromatography with dialysis following each chromatography step. Purity was assessed via sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and concentration determined by the difference in A572 of both the oxidized and the reduced form of the protein.

Formation of Protein Charge Ladders

Protein charge ladders of truncTtRp were produced as previously described for other metalloproteins.9,10 Briefly, truncTtRp was washed via centrifugal filtration (5,000 × g, 4 °C) into 100 mM HEPBS (pH 9.0) to a final protein concentration of approximately 50 μM. Neat acetic anhydride was diluted to 100 mM in 1,4-dioxane. 4 mol equiv of acetic anhydride was added to produce a protein charge ladder. The amount of acetylation was confirmed through electrospray ionization-mass spectrometry (ESI-MS). The acetylated protein was then washed in the running buffer for capillary electrophoresis (20 mM MES, pH 5.5–10.6).

Identification of Lysine Sites Acetylated with Tryptic Fragments and LC/MS/MS

Tryptic fragments of acetylated protein were generated as previously described.37 Prior to proteolysis, truncTtRp was reduced with dithiothreitol (DTT, 2 mM) at room temperature for 1 h. Trypsin Gold (Promega, WI, USA) was added at a molar ratio of 1:20 trypsin to truncTtRp and incubated at 37 °C for 24 h in 50 mM Tris-HCl buffer (pH 8.8). Following digestion this sample was sequenced with LC-MS/MS (LTQ LX/Orbitrap Q Exactive Focus Thermo Scientific).

Proteomic analysis of the MS/MS spectra was performed using SeQuest. Acetylation (+42 Da) on lysine, histidine, and cysteine residues were queried. Peptide spectrum matches (PSMs) were validated with false discovery rate confidence thresholds of 0.01–0.05. PSMs of high and medium confidence were accepted. PSM filtering also considered parameters: Xcorr, ΔCn, and precursor mass accuracy.

Amide Hydrogen/Deuterium Exchange LC-ESI-MS

H/D exchange was performed as previously described.37 Briefly, isotopic exchange was initiated by diluting 100 μL of protein into 900 μL of 99.9% D2O (Cambridge Isotope Laboratories, Inc., Tewksbury, MA, USA). H/D exchange was conducted at 22 °C and pHread = 7.4. 50 μL aliquots of sample were taken and flash frozen at 0 and 60 min. The remaining solution was then placed on a heat block at 90 °C for 5 min to produce a perdeuterated sample to quantify back exchange. Mass spectrometric analysis was completed using a Thermo Scientific Orbitrap Q Exactive Focus. Each time point of truncTtRp was then diluted, and 950 μL of chilled 2% formic acid were immediately loaded on to a desalting column (Michrom BioResources, Inc., Auburn CA, USA) and eluted with a 70:28:2% (v/v) mixture with acetonitrile, water, and formic acid. The desalting apparatus was kept on ice during each analysis. The mass measurements were completed in approximately 3 min from time of thawing (1 min sample preparation, 2 min data collection).

Capillary Zone Electrophoresis

Capillary electrophoresis experiments were performed on a Beckman P/ACE MDQ instrument fitted with a bare fused-silica capillary. All electrophoresis experiments were run at 29 kV in 20 mM MES (pH 5.5–10.6). The capillary was maintained at 22 °C via a cooling jacket (to prevent joule heating). Initial conditioning of the capillary was conducted by washing with 0.1 M HCl (10 min), MeOH (10 min), Milli-Q water (10 min), 0.1 M KOH (10 min), and running buffer (30 min). Between electrophoresis experiments, the capillary was reconditioned via washing with a running buffer (5 min). Dimethylformamide (DMF) was added to each sample as a neutral marker for electroosmotic flow. Oxygen was removed from running buffers for experiments in reducing conditions via nitrogen bubbling for 2 h as previously described.10 Protein solutions were reduced with ∼100 mol equiv of sodium dithionite (5 mM). The reduction of Fe in truncTtRp was verified by UV–vis as previously described.19

Theoretical Determination of Zred of TruncTtRp

The charge of truncTtRp in the reduced state was determined using averaged experimentally determined pKa values of all ionizable residues (i.e., Asp (3.43), Glu (4.14), His (6.45), Tyr (10.98), Lys (10.68), C-term (3.16), N-Term (7.64)).40 The theoretical net charge of truncTtRp was then calculated at every 0.1 pH unit from 5.5 to 11 using the Henderson–Hasselbalch equation and adding the sum of the positive and negative residues. The reduced form of the truncTtRp was chosen, as both active site histidine residues are always protonated from pH 5.5 to 11.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/jacs.3c03006.Figures and tables including MS/MS spectra identifying lysine acetylation; replicate electropherograms and plots of electrophoretic mobility vs acetylation of truncTtRp charge ladders across pH 5.5–10.6; the change in mobility imparted by each acetylation across pH; location of charged residues within 20 Å of [2Fe-2S] cluster; plot of redox potentials and ΔZET across pH; molecular weights of unacetylated and acetylated truncTtRp in 90% D2O. (PDF)

Supplementary Material

ja3c03006_si_001.pdf

This research was supported by grants from the National Science Foundation (CHE: 2203441) and the Welch Foundation (AA-1854 to BFS; W-0031 to Trinity Chemistry Department).

The authors declare no competing financial interest.

Acknowledgments

The authors would like to thank Matthew Guberman-Pfeffer for helpful discussions.
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