Figure 1.

Steady-state and optical parameters of S5H. A buffered solution of S5HR (0.09 μM), S5HF (1.84 μM), and S5HH (0.23 μM), was reacted with a solution of NADH (742 μM) and salicylate (1–50 μM) using a stopped-flow spectrophotometer (2 mm pathlength, concentrations after mixing). Solutions were prepared in air-saturated 50 mM HEPES, 20 mM NaCl, 5 % glycerol, pH 8.0 buffer (~250 μM O₂) at 23 °C. The slope of the initial portion of the time course was determined and converted into an initial velocity using the extinction of NADH at 340 nm, pH 8 corrected for the contribution of the gentisate product at this wavelength and pH (difference ε = 4300 mM⁻¹ cm⁻¹). The solid line is a fit to a hyperbolic expression. Inset: UV-visible spectra of oxidized S5HH (black) (178 μM) and reduced S5HH (red) (178 μM, 5.5 mM sodium dithionite) in standard buffer at 23 °C. Spectra were recorded under anaerobic conditions. The calculated Δε₇₀₀ is 198 M⁻¹cm⁻¹, a value used in data analysis below.