THE EXCHANGE OF HYDROGEN-IONS AND OF WATER-MOLECULES NEAR THE ACTIVE-SITE OF CYTOCHROME-C

Three-pulse electron-spin-echo envelopes have been measured for frozen solution samples of horse heart ferric cytochrome c both before and after exchange against D20. The deuterium modulation pattern was enhanced relative to modulation patterns due to coupled 14N nuclei by dividing the envelope obtained with the nondeuterated sample into the envelope obtained with the deuterated sample. Measurements of the modulation depth were made by fitting single cycles of a decaying waveform having the free deuterium NMR period to early portions of the experimental quotient waveform. The observed modulation depth was compared with the depth calculated theoretically from X-ray crystallographic data. The good agreement obtained in this comparison demonstrates the reliability of the electron-spin-echo method as a means of studying the distance and/or distribution of exchangeable hydrogen nuclei in the vicinity of sufficiently well-characterized protein metal-ion active sites. © 1990.