Pro k software

Flow-flash experiments were performed to study the single-turnover oxidation kinetics of CytcO as described in [41 (link), 42 (link)]. Briefly, the purified CytcO was transferred to a Thunberg cuvette and air was exchanged for N₂. The sample was fully reduced by addition of 2 mM ascorbate and 1 mM phenazine methosulfate (PMS). The N₂ atmosphere was then exchanged for CO. Absorbance spectra were acquired on a Cary 4000 spectrophotometer (Agilent) to monitor the redox state of CytcO and the binding of CO to heme a₃. The kinetic measurements were done using a flow-flash instrument (Applied Photophysics). The delay time between mixing and photo-dissociation of CO was 200 ms and the cuvette path length was 1.00 cm (for other conditions, see the Figure legend of Figure 4). The kinetic data were analyzed using the Pro-K software (Applied Photophysics).

The rate of oxygen dissociation from Ngb-H64Q-CCC was determined by ligand replacement. The experiments were carried out at 25 °C in an Applied Photophysics SX-20 stopped-flow spectrophotometer with a diode array detector (Applied Photophysics Ltd.) contained in an anaerobic glove box (Coy Laboratory Products). 100 mM sodium phosphate buffer, pH 7.4 was used for all the solutions. The Ngb samples were reduced by excess sodium dithionite in the glove box and the deoxy-Ngb was run through a gravity size-exclusion column (Econopac 10DG, BioRad) to remove the excess of reductant. The protein was mixed with air-saturated buffer ([O₂] ≈ 260μM) in 1:4 (v/v) or 1.5:1 (v/v) ratios to form quantitatively the Ngb-oxy complex and achieve final oxygen concentrations of ≈ 208 μM (1:4) or ≈ 104 μM (1.5/1). This sample was then mixed different concentrations of CO buffer made by mixing CO-saturated buffer ([CO] ≈ 1 mM) and anaerobic buffer in the stopped-flow instrument. The spectrum in the visible range (350–730 nm) was sampled every 1.24 ms for a reaction time of 1 s. Spectral changes were consistent with a decay of the Ngb-oxy complex to form the Ngb-CO species. Traces over the whole spectral range (300–730nm) where fitted simultaneously for each experiment using the Pro-K software (Applied Photophysics Ltd.) to calculate the observed oxygen dissociation rate.

Stopped-flow experiments were performed with an SX20 stopped-flow UV-vis spectrometer (Applied Photophysics) with a 1-cm path length cell equilibrated at 4 °C. Apo-HasAp protein solutions were prepared at concentrations ranging from ~ 40 to 400 μM in 200 mM HEPES buffer, pH 7.0, using a 280-nm molar extinction coefficient, ε₂₈₀, of 27 mM⁻¹cm⁻¹. Hemin was dissolved in 10 mM NaOH and diluted to a final concentration of 10 μM just before the stopped-flow experiments using an ε₃₈₅ of 58.4 mM⁻¹cm⁻¹. After each measurement, remaining pre-mixed solutions were recovered from the stopped-flow apparatus to confirm the protein and hemin concentrations. The time-resolved spectra were examined by global analysis using a Marquardt-Levenberg algorithm (Pro-K software, Applied Photophysics). Reported rate constants are the average of at least three different rapid mixing experiments.