Resonance energy transfer studies using a pyrene-labeled phospholipid deriv
ative 1-palmitoyl-2-[10(pyren-1-yl)decanoyl]-sn-glycero-3-phosphoglycerol (
donor) and the heme (acceptor) of cytochrome c (cyt c) have indicated that
ATP causes changes in the conformation of the lipid-bound protein (Rytomaa,
M., Mustonen, P., and Kinnunen, P. K. J. (1992) J. Biol. Chem. 267, 22243-
22248). Accordingly, after binding cyt c via its so called C-site to neat p
hosphatidylglycerol liposomes (mole fraction of PG = 1.0) has commenced, fu
rther quenching of donor fluorescence is caused by ATP, saturating at 2 mM
nucleotide. ATP-induced conformational changes in liposome-associated cyt c
could be directly demonstrated by CD in the Soret band region (380-460 nm)
. The latter data were further supported by time-resolved spectroscopy usin
g the fluorescent cyt c analog with a Zn2+-substituted heme moiety. A high
affinity ATP-binding site has been demonstrated in cyt c (Craig, D. B., and
Wallace, C. J. A. (1993) Protein Sci. 2, 966-976) that is compromised by r
eplacing the invariant Arg(91) to norleucine. Although no major effects on
conformation and function of cyt c were concluded due to the modification,
a significantly reduced effect by ATP on the lipid-bound [Nle(91)]cyt c was
evident, implying that this modulation is mediated via the Arg(91)-contain
ing binding site.