Supplemental Data: Cysteine reactivity and thiol-disulfide interchange pathways in AhpF and AhpC of the bacterial alkyl hydroperoxide reductase

METHODS Spectral and Anaerobic Measurements. All absorbance spectra and anaerobic titrations employed the thermostatted Milton Roy Spectronic 3000 diode array spectrophotometer. The oxidoreductase and DTNB assays were performed on a thermostatted Applied Photophysics DX.17MV stopped-flow spectrofluorometer. The molar extinction coefficients of the proteinbound FAD at 448 or 449 nm were determined by release of the flavin cofactor with 4.0 M guanidine hydrochloride and quantitation of the corresponding free FAD (2). Anaerobic titrations were carried out essentially as previously described (3). NADH titrating solutions were prepared in buffer bubbled for 20-30 min with oxygen-free nitrogen, loaded into the titrating syringe, and standardized prior to each experiment. An oxygenscrubbing system consisting of protocatechuate 3,4-dioxygenase and protocatechuic acid was included in all NADH titration experiments. Spectral data were not corrected for dilution, which was <3% overall. Transhydrogenase and oxidase assays. Tranhydrogenase assays were carried out at 25°C in 1 mL of solution containing 150 μM each of NADH and AcPyAD, 50 mM Tris-HCl at pH 8.0, 0.5 mM EDTA, 100 mM ammonium sulfate, and 10-20 pmol of AhpF monitoring the gain in absorbance at 390 nm due to the formation of AcPyADH (2).