M. Aki et al., A new measurement system for UV resonance Raman spectra of large proteins and its application to cytochrome c oxidase, J PHYS CH B, 104(46), 2000, pp. 10765-10774
A new type of ultraviolet resonance Raman (UVRR) measurement system suitabl
e to a limited amount of large protein samples is proposed and the results
from its application to bovine cytochrome c oxidase (CcO) is presented. To
minimize the sample damage caused by high-flux UV laser illumination and to
reject visible fluorescence from the sample, frequency-doubling of a mode-
locked Ar+ ion laser and a solar blind multichannel detector were employed,
respectively. A new spinning cell was designed so that the sample solution
could be stirred during spinning of the cell. Combination of all these dev
ices resulted in successful observation of high quality UVRR spectra of CcO
excited at 244 nm. The RR bands of tryptophan and tyrosine residues domina
ted the observed spectra, while an extra band appeared at 1656 cm(-1). The
frequency of the extra band as well as those of all other bands were unalte
red by the redox change of metal centers and ligand binding to heme a(3). D
eprotonation of a tyrosine residue(s) with a low pK(a) value was detected f
or the resting state at pH 9.1. Examination of all possible assignments led
us to conclude that the extra band arose from the linoleoyl side chain of
phospholipids and its intensity suggested the presence of 21 linoleoyl grou
ps per CcO molecule.