MB, EPR, and UV–vis spectra
were collected and analyzed as described.23 (link) MB spectra were collected using a MS4 WRC spectrometer (SEE Co.,
Edina MN), calibrated using α-Fe foil at room temperature. Spectra
were analyzed and simulated using WMOSS software. EPR spectra were
collected at the University of Texas at Arlington on an X-band EMX
spectrometer (Bruker Biospin Corp., Billerica, MA) equipped with a
bimodal resonator (4116DM) and an Oxford Instruments ESR900 cryostat.
Signals were integrated using a custom Matlab (Mathworks.com) program.
Spin concentrations were calculated as described24 (link) using 1.00 mM CuSO4-EDTA as the standard.
UV–vis spectra of whole cells were collected on a Hitachi
U3310 spectrometer with a Head-on photomultiplier tube. Spectra were
simulated using OriginPro software as described.23 (link) Packed-cell samples were diluted 3-fold with ddH2O and analyzed in a 10 mm path length quartz UV–vis cuvette
(Precision cells). After collecting MB spectra, isolated mitochondria
were thawed anaerobically in a glovebox (≤5 ppm of O2, MBraun, Labmaster), diluted 3-fold with SH buffer (0.6 M Sorbitol,
0.02 M HEPES, Fisher Scientific, pH 7.4), and transferred to a 2 mm
path length quartz UV–vis cuvette (Precision cells). The cuvette
was sealed with a rubber septum and brought out of the glovebox and
immediately analyzed by UV–vis.
were collected and analyzed as described.23 (link) MB spectra were collected using a MS4 WRC spectrometer (SEE Co.,
Edina MN), calibrated using α-Fe foil at room temperature. Spectra
were analyzed and simulated using WMOSS software. EPR spectra were
collected at the University of Texas at Arlington on an X-band EMX
spectrometer (Bruker Biospin Corp., Billerica, MA) equipped with a
bimodal resonator (4116DM) and an Oxford Instruments ESR900 cryostat.
Signals were integrated using a custom Matlab (Mathworks.com) program.
Spin concentrations were calculated as described24 (link) using 1.00 mM CuSO4-EDTA as the standard.
UV–vis spectra of whole cells were collected on a Hitachi
U3310 spectrometer with a Head-on photomultiplier tube. Spectra were
simulated using OriginPro software as described.23 (link) Packed-cell samples were diluted 3-fold with ddH2O and analyzed in a 10 mm path length quartz UV–vis cuvette
(Precision cells). After collecting MB spectra, isolated mitochondria
were thawed anaerobically in a glovebox (≤5 ppm of O2, MBraun, Labmaster), diluted 3-fold with SH buffer (0.6 M Sorbitol,
0.02 M HEPES, Fisher Scientific, pH 7.4), and transferred to a 2 mm
path length quartz UV–vis cuvette (Precision cells). The cuvette
was sealed with a rubber septum and brought out of the glovebox and
immediately analyzed by UV–vis.
