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 pKa 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?

“…[18,19] In contrast, the capillary electrophoresis of a" protein charge ladder" requires nanoliters of protein solution, at micromolar concentration and < 10 minutes per experiment. [1,2,4,6] We estimate that the net charge of only about 0.1 %o ft he approximately7 0000 unique proteins currently in the Protein Data Bank have been measured in their folded state at pH ¼ 6 pI. Of course,i soelectricp oints can be measured for thousands of proteins in as ingle experiment, but isoelectric points are poor expressions of net charge.T he pI of ap rotein represents only one value of Z at one value of pH (i.e., Z = 0a tp H= pI).…”

“…This Concept article discusses how an overlooked tool in analytical chemistry,t he "protein chargel adder," [1][2][3][4] is beginning to provide new quantitative information about the fundamental electrostatic properties of metalloproteins. [5][6][7] When analyzed with capillary zone electrophoresis (CZE or CE), a"protein charge ladder" (Figure 1A,B) is one of the few tools that can rapidly quantify the net electrostatic charge(Z)ofafolded pro-tein in solution. Measuring this overlooked, fundamental parameter can quantify the driving forces of biochemical reactions, especially those involving metalloproteins.…”

“…Measuring this overlooked, fundamental parameter can quantify the driving forces of biochemical reactions, especially those involving metalloproteins. Here, we will discusst wo recent applications of "proteinc harge ladders" from our laboratory:1 )quantifying how the net chargeo f three metalloproteins (azurin, cytochrome c, and myoglobin) are affected by single electron transfer (ET); [6] and 2) quantifying how the net charge of superoxide dismutase (SOD1) is affected by the binding of Cu 2 + and Zn 2 + ;s olvent pH;a nd nonisoelectricm utationst hat cause amyotrophic lateral sclerosis (ALS). [5,7] As ubset of ALS-linkedS OD1 mutationsa re suspected to trigger SOD1 aggregation, relatedt oA LS pathogenesis, by minimizing electrostatic repulsions between SOD1 polypeptides and by diminishingt he affinity of SOD1 to coordinate Cu 1 + /2 + and Zn 2 + .…”

“…[1,4] Deoxyribonuclease and ovalbumin, for example, have identical isoelectricp oints of pI = 5.1, but the formal and measured net charge of both proteins differ by approximately 7u nits at pH 8.4. [4] The systematic absence of experimentally determinedv alues of Z has likely impeded ar igorous understanding of most chemicalp rocesses in which proteinsa re involved including aggregation and self-assembly, [20][21][22][23][24][25][26] ligand binding, [27][28][29][30][31][32][33][34] catalysis, [35][36][37][38][39] electron transfer, [3,6,[40][41][42][43][44][45][46][47] protein crystallization, [14,48] analytical separation, [49,50] and protein engineering. [51][52][53][54][55][56] It is tempting to assume that the formal net chargeo faprotein predicted from generalized residue pK a values (Z seq )issosimilar to the actual net charge that any difference is irrelevant, and the isoelectric point tells us all we need to know about ap rotein's net charge.…”

“…[1] These studies and others have shown that the effective net charge of ap rotein can be different in sign and magnitude (by > 10 units) than values predicted from formal pK a values. [2,[4][5][6][7]78] Climbing a"Protein Charge Ladder" for a Better View of Electron Transfer A" protein chargeladder" is an electrophoretic array of variably charged (but similarly shaped) proteins. Protein chargel adders can be prepared by successively acetylating (neutralizing) surface Lys-e-NH 3 + with acetic anhydride( Figure 1A-C, 2A,B).…”