Table 1.

Summary of the pK_a estimation

Calculated pK_a values for absorption changes in the pH titration in Fig. 5. Protein variant, 15Z/15E configuration, and absorption peak wavelength utilized for the fitting are shown. Errors are reported as standard deviations of the mean for the fitted values. We utilized the pK_a value with smaller errors for the transition. For example, pK_a of ^15ZPg/^15ZPo conversion of C73A 15Z state can be calculated as 5.8 ± 0.8 from the decrease of ^15ZPg at 550 nm and 6.4 ± 0.2 from the increase of ^15ZPo peak at 600 nm. We described 6.4 ± 0.2 as reliable pK_a values for the ^15ZPg/^15ZPo conversion in the main text.

Protein variant and D-ring configuration	pKa	Absorption peaks for pKa calculation
WT 15E	9.0 ± 0.1	15EPo (600 nm)
	9.4 ± 0.4, 9.6 ± 0.4	15EPb (420 nm)
	10.0 ± 0. 1	15EPg (530 nm)
C73A 15Z	5.8 ± 0.8	15ZPg (550 nm)
	6.4 ± 0.2	15ZPo (600 nm)
C73A 15E (with minor 15Z)	6.7 ± 0.7, 9.9 ± 0.3	15EPg (530 nm)
	6.2 ± 0.7, 9.4 ± 0.1	15EPo (600 nm)
D71A 15Z	6.0 ± 0.2	15ZPo (610 nm)
	9.7 ± 0.1	15ZPb (420 nm)
	9.7 ± 0.2	15ZPg (550 nm)
D71A 15E	10.6 ± 0.9	15EPg (550 nm)
	8.8 ± 0.1	15EPo (610 nm)