Table 2.

Impact of putative uncoupling agents on sulfite to oxygen stoichiometry

Enzyme	Substrate	SO32−:O2 ratio*†
		0 ascorbate	20 μM ascorbate	1 mM ascorbate	Average‡	20 mM buffer	200 mM buffer
Wild-type	H	0.97 ± 0.04	0.97 ± 0.04	1.00 ± 0.04	0.99 ± 0.07	≈ 1§	≈ 1§
	D	0.61 ± 0.04	0.74 ± 0.06	0.70 ± 0.04	0.70 ± 0.10	0.91 ± 0.06	0.72 ± 0.05
F159L	H	0.67 ± 0.08	0.73 ± 0.04	0.74 ± 0.01	0.72 ± 0.10	0.88 ± 0.02	0.65 ± 0.03
	D	ID¶	ID¶	0.17 ± 0.01	0.17 ± 0.02	0.22 ± 0.02	0.13 ± 0.01
F159V	H	—	—	—	—	0.79 ± 0.02	0.38 ± 0.02
F159A	H	—	—	—	—	0.55 ± 0.04	0.29 ± 0.02
F159G	H	ID¶	ID¶	0.21 ± 0.02	0.22 ± 0.03	0.52 ± 0.04	0.12 ± 0.05

H, 1,1-[¹H₂]; D, 1,1-[²H₂]-2-methylaminoethane-1-sulfonic acid.

*All experiments were performed at ambient O₂ and 30 °C. Experiments in which ascorbate was varied were performed in 50 mM bis-Tris pH 6.2 at the indicated concentration of ascorbate. Experiments in which buffer was varied were performed in the indicated concentration of bis-Tris pH 6.2 with 200 μM ascorbate. The stoichiometries at 50 mM buffer and 200 μM ascorbate are shown in Fig. 2.

^†Errors reported are 2σ determined from multiple experiments (n = 3–5).

^‡The values reported are the averages of the values at all ascorbate concentrations.

^§These values were not determined in triplicate, but single measurements were within experimental error of unity. In light of an absence of an effect of the 10-fold increase in buffer concentration on the wild-type behavior, the decision was made to allow the ionic strength to vary in subsequent experiments.

^¶Indeterminate: the enzymes did not undergo sufficient turnovers to allow reproducible measurements of sulfite.