Influence of side chain length and acidity on H⁺ transport. Proton pumping (traces on right) was compared for exchangers carrying the side chains indicated. Nominal aqueous pK_a values, reported to ±0.5 unit, are taken from model compounds or calculated in SciFinderScholar software. Non-natural side chains were introduced on Cys-less background by reaction of the E203C mutant with IAA (Cys-1), FIAA (Cys-2), 2-carboxylethyl methanethiosulfonate (Cys-3), or 2-sulfonatoethyl methanethiosulfonate (Cys-4). Reactions were >90% complete for all reagents except FIAA, which reacted to only to 70% completion; any unmodified Cys mutant, being inactive (Fig. 1), does not contribute to the signal. Scale bars represent 4 nmol/µg protein, 10 s.