The Proton in Biochemistry: Impacts on Bioenergetics, Biophysical Chemistry, and Bioorganic Chemistry

Abstract

The proton is the smallest atomic particle, and in aqueous solution it is the smallest hydrated ion, having only two waters in its first hydration shell. In this article we survey key aspects of the proton in chemistry and biochemistry, starting with the definitions of pH and pK _a and their application inside biological cells. This includes an exploration of pH in nanoscale spaces, distinguishing between bulk and interfacial phases. We survey the Eigen and Zundel models of the structure of the hydrated proton, and how these can be used to explain: a) the behavior of protons at the water-hydrophobic interface, and b) the extraordinarily high mobility of protons in bulk water via Grotthuss hopping, and inside proteins via proton wires. Lastly, we survey key aspects of the effect of proton concentration and proton transfer on biochemical reactions including ligand binding and enzyme catalysis, as well as pH effects on biochemical thermodynamics, including the Chemiosmotic Theory. We find, for example, that the spontaneity of ATP hydrolysis at pH ≥ 7 is not due to any inherent property of ATP (or ADP or phosphate), but rather to the low concentration of H⁺. Additionally, we show that acidification due to fermentation does not derive from the organic acid waste products, but rather from the proton produced by ATP hydrolysis.

Keywords: acid catalysis; acidity; aqueous solution; diffusion; enzyme catalysis; pH; proton transfer; proton transport.

FIGURE 1

(A) The Eigen cation: H⁺, the large red bolded H on the right is covalently bonded to a single water in its first hydration shell. The three hydrogens in the resulting H₃O⁺ (red) each form hydrogen bonds with a black water (H₂O•) forming the second hydration shell. Bonds are not drawn to scale: O-H covalent bonds (red) have 0.96 Å bond lengths, whereas the H₂O•H hydrogen bonds above are 1.4–1.7 Å long (Asthagiri et al., 2005) (Markovitch et al., 2008). (B) The Zundel cation: The central large red bolded H⁺ is shared equally between two inner shell waters (red), to form an H₅O₂ ⁺ dihydrate that features two symmetrical, unusually short and strong H-bonds. The symmetric trans conformation drawn here is the ground state in bulk water. (C) The Reed/Stoyanov cation: The Zundel dihydrate is expanded to include four black waters (H₂O•) in a second hydration shell; the two central H-bonds in the dihydrate core are slightly longer and weaker than in the Zundel dihydrate (Reed, 2013). The asymmetric cis “sawhorse” conformation drawn here is believed to be the ground state at the hydrophobic phase/water interface.

FIGURE 2

Water-catalyzed 1,3-tautomerization.

FIGURE 3

De Grotthuss proton hopping mechanism, with a chain of three waters, proton input from the left, and output to the right.

FIGURE 4

Simple proton wire comprising two water molecules and two carboxylate side chains.

FIGURE 5

Chymotrypsin catalytic mechanism.

FIGURE 6

pH dependence of chymotrypsin-catalyzed hydrolysis of N-acetyl-L-tryptophanamide at 25°C. Data adapted from (Himoe et al., 1967) are fit to Eq. 13 with best-fit parameters: pK_a,lo = 6.67 ± 0.11, pK_a,hi = 9.57 ± 0.08, v_0,opt.pH = 0.82 ± 0.03 μM/s, R ² = 0.93.

FIGURE 7

Structures of ATP, ADP, and inorganic phosphate.

FIGURE 8

∆G for ATP hydrolysis as a function of pH; I = 0.25 M, [Mg²⁺] = 1 mM, T = 25°C. Data are taken from (Alberty and Goldberg, 1992) and (Méndez and Cerdá, 2016). Measured values are between pH 3.5–6.5 (black circles), 7–10 (purple circles), and pH 0 (open blue squares). Best-fit line for pH ≥ 7 points has intercept = 0.54 ± 0.17 kcal/mol, slope = −1.297 ± 0.020 kcal/mol/pH unit, R ² = 0.9986.