Direct Measurement of Charge Regulation in Metalloprotein Electron Transfer

Abstract

Determining whether a protein regulates its net electrostatic charge during electron transfer (ET) will deepen our mechanistic understanding of how polypeptides tune rates and free energies of ET (e.g., by affecting reorganization energy, and/or redox potential). Charge regulation during ET has never been measured for proteins because few tools exist to measure the net charge of a folded protein in solution at different oxidation states. Herein, we used a niche analytical tool (protein charge ladders analyzed with capillary electrophoresis) to determine that the net charges of myoglobin, cytochrome c, and azurin change by 0.62±0.06, 1.19±0.02, and 0.51±0.04 units upon single ET. Computational analysis predicts that these fluctuations in charge arise from changes in the pK a values of multiple non‐coordinating residues (predominantly histidine) that involve between 0.42–0.90 eV. These results suggest that ionizable residues can tune the reactivity of redox centers by regulating the net charge of the entire protein–cofactor–solvent complex.

What Are We Missing by Not Measuring the Net Charge of Proteins?

“…For example, the accurate measurement of Z from the Gibbs–Donnan effect (through ultracentrifugation) requires milliliters of protein solution at high purity (>95 %), high protein concentration (near millimolar), and hours per experiment . In contrast, the capillary electrophoresis of a “protein charge ladder” requires nanoliters of protein solution, at micromolar concentration and <10 minutes per experiment …”

“…The utility of “protein charge ladders” and their unique capabilities in determining protein net charge has been reviewed . These studies and others have shown that the effective net charge of a protein can be different in sign and magnitude (by >10 units) than values predicted from formal p K a values …”

“…A “protein charge ladder” is an electrophoretic array of variably charged (but similarly shaped) proteins. Protein charge ladders can be prepared by successively acetylating (neutralizing) surface Lys‐ ϵ ‐NH 3 + with acetic anhydride (Figure A–C, A,B) . Each “rung” or Lys‐acetyl derivative differs in net charge by approximately one unit (typically ≈0.9 units, but ranging from ≈0.5 to ≈0.9 depending on the pH, due to charge regulation) .…”

“…This Concept article discusses how an overlooked tool in analytical chemistry, the “protein charge ladder,” is beginning to provide new quantitative information about the fundamental electrostatic properties of metalloproteins . When analyzed with capillary zone electrophoresis (CZE or CE), a “protein charge ladder” (Figure A,B) is one of the few tools that can rapidly quantify the net electrostatic charge ( Z ) of a folded protein in solution.…”

“…Measuring this overlooked, fundamental parameter can quantify the driving forces of biochemical reactions, especially those involving metalloproteins. Here, we will discuss two recent applications of “protein charge ladders” from our laboratory: 1) quantifying how the net charge of three metalloproteins (azurin, cytochrome c, and myoglobin) are affected by single electron transfer (ET); and 2) quantifying how the net charge of superoxide dismutase (SOD1) is affected by the binding of Cu 2+ and Zn 2+ ; solvent pH; and non‐isoelectric mutations that cause amyotrophic lateral sclerosis (ALS) . A subset of ALS‐linked SOD1 mutations are suspected to trigger SOD1 aggregation, related to ALS pathogenesis, by minimizing electrostatic repulsions between SOD1 polypeptides and by diminishing the affinity of SOD1 to coordinate Cu 1+/2+ and Zn 2+ …”