For cw EPR measurements, proteins were concentrated to 100 to 400 μM and supplemented with 20% (w/v) glycerol as a cryoprotectant. Samples of 60 μl were filled into 3-mm quartz tubes and flash-frozen in liquid nitrogen. Samples containing Cu1+ were kept anaerobically, and the quartz tubes were sealed under vacuum after freezing.

Cw EPR spectra were recorded in independent duplicates on an ElexSys E500 spectrometer (Bruker BioSpin, Rheinstetten, Germany) at X-band (~9.4 GHz) using an SHQ resonator and an ESR900 helium flow cryostat (Oxford Instruments, Oxfordshire, UK). All spectra were baseline-corrected by a first-order polynomial, the magnetic field offset was corrected using diphenyl-1-picrylhydrazyl (Sigma-Aldrich, Buchs, Switzerland) as a reference, and the magnetic field axis was rescaled to display all spectra at a common microwave frequency of 9.5 GHz. The spectra were recorded at 40 K using microwave powers of 8 to 32 μW (nonsaturating conditions). The magnetic field modulation amplitude was set to 0.3 mT with a frequency of 100 kHz for lock-in amplification with a conversion time and time constant of 81.92 ms. EPR spectra were analyzed using MATLAB and EasySpin (62) and scaled to correct for differences in protein concentration in the samples. EPR spectra of mixtures were decomposed by least-squares fitting of weighting factors of individual experimental spectra such that the weighted sum corresponded to the experimental spectrum of the respective mixture.

Pulsed EPR distance measurements between Cu2+ ions were carried out by ultra-wideband four-pulse DEER (46, 47) at ca. 9.3 GHz and a temperature of 10 K using a homebuilt X/Q band pulse EPR spectrometer (63) equipped with an MS-3 resonator (Bruker BioSpin, Rheinstetten, Germany) and 40-μl sample in 3-mm quartz tubes (as above). Observer pulses were all rectangular 12-ns pulses applied 50 MHz below the spectral maximum with an initial interpulse delay of 200 ns and incremented in eight steps of 8 ns to average out nuclear modulations. The pump pulse was a hyperbolic secant pulse of order 6 with a length of 64 ns and an excitation bandwidth of 450 MHz, was applied with an offset of +125 MHz, and included nonlinear compensation for resonator and microwave amplifier profiles (63). The pump pulse was stepped by 8 ns, and the repetition rate of the pulse sequence was 100 Hz. The data were analyzed using DeerAnalysis 2018 (64).

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