10 mm quartz cuvette

To collect CD spectra for both wild-type TsaM and variants TsaM, we capitalized on the CD spectra collection methods that were used in our previous work^{21 (link),48 (link)}. These methods use 350 µL samples of 5–10 µM wild-type TsaM and TsaM variants, a 10 mm quartz cuvette (Hellma), and a Jasco J-1500 CD spectrometer.

Stock solutions of wild-type RexT were diluted to 10 µM using a buffer containing 50 mM HEPES (pH = 8.0) and 150 mM NaCl. H₂O₂ was added into the protein solution with a final concentration of 1:1 or 2:1 to protein concentration. This mixture was incubated at room temperature for 5, 15, and 30 min and the CD spectrum of each sample was taken subsequently. For each CD measurement, 400 µL of the diluted RexT -H₂O₂ solution was transferred into a 10 mm quartz cuvette (Hellma). The CD spectra were recorded using a Jasco J-1500 CD spectrometer with 0.1 nm data pitch and 20 nm/min scan speeds. The baseline was measured with the same buffer used to do dilution (50 mM HEPES (pH = 8.0) and 150 mM NaCl). Each sample spectrum is an average of five cumulative spectra.

The quality of proteins used in this study was assessed based on the guidelines established by ARBRE-MOBIEU and P4EU (https://arbre-mobieu.eu/guidelines-on-protein-quality-control/). Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was used to check the purity of proteins. UV/visible spectra from 220–340 nm were recorded on a Nanodrop ND-100 spectrometer (PEQLAB, Erlangen, Germany) to ensure the absence of nucleic acids and aggregation. In order to determine concentrations the absorbance at 280 nm was measured in a 10 mm quartz cuvette (Hellma, Müllheim, Germany) on a Biospectrometer basic (Eppendorf, Hamburg, Germany). Identity was confirmed by peptide mass fingerprinting (Department of Biochemistry, University of Bayreuth, Germany) and homogeneity was ensured by analytical gel filtration using a Superdex 75 or a Superdex 200 10/300 GL column (GE Healthcare, Munich, Germany). Circular dichroism (CD) spectroscopy (1 mm quartz cuvette; J-1100, JASCO, Pfungstadt, Germany) was performed to assess the folding state (the folding state of unlabeled λQ and λQ^Δ36 was additionally checked by recording one dimensional ¹H-NMR spectra).

CS activity was determined in isolated mitochondria as described previously [40 (link)]. A frozen subsample of the isolated mitochondria dissolved in MiR05 was thawed on ice. A reaction medium containing 100 μl of 0.1 mM 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB), 25 μl of 10% Triton X-100, 50 μl of 10 mM oxaloacetate, 25 μl of 12.2 mM acetyl coenzyme A, and 790 μl purified water was mixed and preheated at 30 °C for 5 min. Subsequently, 10 μl of the isolated mitochondria was added to the reaction medium and transferred into a 10-mm quartz cuvette (Hellma^® Analytics, Muellheim, Germany). CS activity was assessed at 412 nm using a GENESYS 5 spectrophotometer (Spectronic via Thermo Fisher Scientific, Waltham, MA, USA) and normalized to protein content. Measurements were performed in duplicate.