Itc 503 temperature

X-band EPR measurements were performed
on a Bruker ELEXYS E500 spectrometer equipped with a SuperX EPR049
microwave bridge and a cylindrical TE₀₁₁ ER 4122SHQE cavity
in connection with an Oxford Instruments continuous flow cryostat.
Measuring temperatures were achieved using liquid helium flow through
an ITC 503 temperature controller (Oxford Instruments). The Xepr software
package (Bruker) was used for data acquisition and processing. EasySpin
software version easyspin-6.0.0-dev.51 was used for spectral simulation
and fitting.^{39 (link)} EPR samples of E252V and
E289D were prepared in 100 mM Tris–HCl, pH 8.0 under a neat
argon atmosphere and either directly flash-frozen (“as prepared”)
or flushed with H₂ or CO gas inside the EPR tube for an
hour prior to freezing (“H₂- or CO- flushed”).
EPR samples incubated with D₂ were prepared in the absence
(4 μL of 1 mM enzyme in 10 mM Tris–HCl, pH 8.0 + 76 μL
100 mM Tris–HCl, pH 8.0) or presence of 95% D₂O
(4 μL of 1 mM enzyme in 10 mM Tris–HCl, pH 8.0 + 76 μL
D₂O; Figure S6). For samples
that were reduced with NaDT, 1 μL of 100 mM stock solution of
NaDT was added into 79 μL of 50 μM enzyme, resulting in
at least 20-fold molar excess of NaDT (Figure S7).