Varian cary4000

Absorbance spectra were collected using a Varian Cary4000 spectrophotometer (Agilent Technologies, Santa Clara, CA, USA) on solutions containing 34 μM hSR, 50 mM TEA pH 8.0, at 20.0 ± 0.5 °C. Fluorescence emission spectra upon excitation at 412 nm were collected using a FluoroMax-3 fluorometer (HORIBA Jobin Yvon, Kyoto, Japan) at 20.0 ± 0.5 °C. The solution for fluorescence measurements contained 2.7 µM hSR in buffer A with 5 mM TCEP. Binding of either glycine or ATP was measured by direct titrations of the protein solution in the range 0–1.2 mM and 0–1.0 mM, respectively. Added volume did not exceed 20% of the initial volume and the spectra were corrected for dilution. Spectra were corrected for buffer contribution. Far-UV circular dichroism spectra (195–260 nm) were collected using a JASCO J-715 spectropolarimeter (Easton, MD, USA). Measurements were performed at 20.0 ± 0.5 °C on a solution containing 4 μM hSR, 20 mM NaH₂PO₄ pH 7.5. Each spectrum is the average of three independent measurements.

GcoB activity was assessed using a continuous colorimetric assay involving cytochrome c as a colorimetric electron acceptor^{35 (link)}. A total of 4.8 nM GcoB (referenced to [FAD]) and 42 µM cytochrome c (from equine heart) were dissolved in buffer (25 mM HEPES, 50 mM NaCl, pH 7.5), 25 °C. 100 µM NADH was then added to initiate the NADH- and GcoB-dependent reduction of cytochrome c. UV/vis spectra (Varian Cary 4000, Agilent) were recorded in the scanning kinetics mode. The increase in absorbance at 550 nm due to reduced cytochrome c was monitored over time, and the specific activity (nmol reduced cytochrome c min⁻¹ nmol GcoB⁻¹) calculated using:^{35 (link)} $$\Delta A_{550}{\mathrm{m}in}^{-1}∕0.021∕\mathrm{m}L\mathrm{r}eaction=\mathrm{s}pecific\mathrm{a}ctivity.$$
For determining steady-state kinetic constants, the above protocol was used as a function of [NADH]. A total of 4.8 nM GcoB (referenced to [FAD]) and 43 µM cytochrome c were dissolved in buffer (25 mM HEPES, 50 mM NaCl, pH 7.5), 25 °C, and the reaction was initiated with the addition of 2.5–200 µM NADH. Initial velocities (v_i) were determined from linear fits to the initial portion of the progress of reaction data, plotted as a function of [NADH], and fit to the Michaelis–Menten Eq. (5) using the KaleidaGraph software: $$v_i=V_{\mathrm{m}ax}\left[S\right]∕\left(K_{\mathrm{M}}+\left[S\right]\right).$$