Itc 502

All refocused echo experiments on the low spin Fe(III) neuroglobin were carried out using a continuous flow helium cryostat (CF935) and a temperature control system (ITC 502) from Oxford Instruments. This was carried out at 6 K using the observer part of the DEER sequence (Figure 1), π/2τ 1 -π -(τ 1 + τ 2 ) -π -τ 2 -echo with pulse lengths t π/2 = 8 ns and t π = 16 ns for standard pulses and t πcomposite1 = 8+16+8 ns (90 90 180 0 90 90 , Levitt [1]) or t πcomposite2 = 8+16+24 ns (90 0 180 180 270 0 , Shaka [23] (link)) for composite pulses, with inter-pulse delays of τ 1 =198 ns and τ 2 = 954 ns.
Refocused echo experiments on MSA236 were carried out at room temperature (291 K) using the same sequence and pulse lengths as for the neuroglobin, with inter-pulse delays of τ 1 =250 ns and τ 2 = 750 ns.
DEER experiments were carried out on MSA236 at room temperature (291 K) using the four-pulse DEER sequence (Figure 1), π/2(f probe )-τ 1 -π(f probe ) The field swept echo experiment on MSA236 was carried out at 291 K using a Hahn echo sequence, π/2 -τ -π -τ -echo, with 7 and 14 ns π/2 and π pulse lengths and inter-pulse delay τ = 250 ns. The field was swept from 3337 mT to 3367 mT in 0.1 mT steps, with pulse frequency of 93.9996 GHz.
Sequence repetition frequency was 5 kHz, averaging 5,000 shots per point.

A Mixture of [Eu(tmh)3(MeOH)2] (0.5 g, 0.7 mmol) and [Tb(tmh)3(MeOH)2] (0.5 g, 0.7 mmol) was dissolved in methanol (30 mL). The dpbp ligand (0.39 g, 0.7 mmol) was added to it. Optical measurements: Emission and excitation spectra were measured with a spectrofluorometer (HORIBA Fluorolog-3). Emission quantum yields were estimated using a spectrofluorometer (JASCO FP-6300) with an integrating sphere unit (JASCO ILF-533). The wavelength dependence of the detector response and beam intensity of the Xe light source for each spectrum were calibrated using a standard light source. Emission lifetimes were measured using the third harmonic (355 nm) of a Q-switched Nd:YAG laser (Spectra-Physics, INDI-50, fwhm = 5 ns, λ = 1064 nm) and a photomultiplier (Hamamatsu Photonics, R-5108, response time < 1.1 ns). The Nd:YAG laser response was monitored with a digital oscilloscope (Sony Tektonics, TDS3052, 500 MHz) synchronized to single pulse excitation. Emission lifetimes were determined from the slopes of logarithmic plots of decay profiles. Temperature-dependent emission spectra and emission lifetimes were measured using a cryostat (Thermal Block Company SA-SB245T) and a temperature controller (Oxford Instruments ITC-502S). Diffuse reflection spectra were recorded using a spectrophotometer (JASCO V-670) with an integrating sphere unit (JASCO ISN-723).