J 725 cd spectropolarimeter

Optical absorption spectra of oxidized WT and M80A human cyt c were measured with a UV-2450 spectrophotometer (Shimadzu) using a 1-cm path-length quartz cell at 25 °C. The extinction coefficients of oxidized monomeric and dimeric M80A cyt c were determined with the prydine hemochrome method [32] . The pH titration of oxidized M80A cyt c was performed from pH 8.74 to 2.75 by a continuous addition of a small amount of 1.0 M HCl to cyt c in 50 mM potassium phosphate. The dilution of cyt c solution with 1.0 M HCl was less than 6% at the final point (pH 2.75), and the intensity of each absorption spectrum was calibrated according to its cyt c concentration change by the dilution. Absorbance at 398.5 nm (isosbestic point between His/H2O 6-coordinate and His 5-coordinate species; pKa = 3.8 [33] ) was plotted against pH, and the pKa value was determined by least-square fitting the data to the Henderson-Hasselbalch equation using the software, Igor Pro 6.0.
Circular dichroism (CD) spectra of oxidized WT and M80A cyt c were measured with a J-725 CD spectropolarimeter (Jasco, Japan) using a 0.1-cm-path-length quartz cell at room temperature.

Circular dichroism (CD) spectra of ferric Met80-modified and unmodified horse cyt c (8 μM) were measured with a J-725 CD spectropolarimeter (Jasco) using a 0.1-cm pathlength quartz cell at 20 °C. Protein solutions in 50 mM potassium phosphate buffer, pH 7.0, were filtered through the 0.45 μm filter (Millipore) before measurements.