Figure 5.

Nitrogen-15 titration curves and analysis. (A, B) Data from 1D ¹⁵N NMR spectra of 12 mM oxidized [U-¹³C,¹⁵N]-His,[¹⁵N]-Leu TtRp collected at 278 K at various pH values. On the basis of data shown in Figures 4 and 5, the lower pK_a value was assigned to His154 and the higher pK_a value was assigned to His134 (Table 2). (C−E) Data from 1D ¹⁵N NMR spectra of ∼5 mM reduced [U-¹³C,¹⁵N]-His,[¹⁵N]-Leu TtRp collected at 298 K at various pH values. (A) Representative ¹⁵N NMR spectra of oxidized TtRp. Peak assignments are shown over the top trace. (B) The pH dependence of the chemical shifts of the peaks assigned to the ¹⁵N^ε2 of His134 (open squares) and the ¹⁵N^ε2 of His154 (filled circles) of oxidized TtRp. Fitting of the data points (curves) yielded His134 pK_a = 9.16 ± 0.02 and His154 pK_a = 7.58 ± 0.01. (C) Representative ¹⁵N NMR spectra of reduced TtRp. The ¹⁵N^ε2 signals of His134 and His154 (denoted by ∗) overlapped between pH 8.14 and 10.48 and disappeared from spectra at higher pH. (D) Correlation between the His154 ¹³C′ and ¹⁵N^ε2 chemical shifts for the first six titration points. Linear regression yielded a correlation coefficient of 0.998. (E) Correlation between His134 C′ and the ¹⁵N^ε2 for the first six points. Linear regression line yielded a correlation coefficient of 0.992. The inset shows the 2D ¹³C′−¹³C^α cross peak assigned to His134 at different pH values (cross peaks move to the left with increasing pH) collected at the pH values corresponding to the top 6 spectra in panel C.