ATP Induces a Conformational Change in Lipid-bound Cytochrome c

Esa K.J Tuominen,Keng Zhu,Carmichael J.A Wallace,Ian Clark-Lewis,Douglas B Craig,Marjatta Rytömaa,Paavo K.J Kinnunen

doi:10.1074/jbc.m100853200

Abstract

Resonance energy transfer studies using a pyrene-labeled phospholipid derivative 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 (Rytömaa, 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 phosphatidylglycerol liposomes (mole fraction of PG = 1.0) has commenced, further 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 using the fluorescent cyt c analog with a Zn(2+)-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 replacing 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)-containing binding site.

Highlights

Resonance energy transfer studies using a pyrenelabeled phospholipid derivative 1-palmitoyl-2-[10(pyren-1-yl)decanoyl]-sn-glycero-3-phosphoglycerol and the heme of cytochrome c have indicated that ATP causes changes in the conformation of the lipid-bound protein
In cyt c part of the high affinity ATP-binding site is constituted by the invariant Arg91 [5, 7,8,9,10], and binding of ATP to this decreases the rate of electron flow through the mitochondrial electron transport chain [9]
The C-site interaction of cyt c is not dissociated by nucleotides, and elevated ionic strength is able to dissociate the protein only if it causes the deprotonation of acidic phospholipids, changing the interaction from C- to A-site

Summary

EXPERIMENTAL PROCEDURES

Materials—1-Palmitoyl-2-[10-(pyren-1-yl)decanoyl]-sn-glycero-3phosphoglycerol (PPDPG) was purchased from K&V Bioware (Espoo, Finland). ATP was included to yield up to 5 mM final concentration, and 5- or 10-␮l aliquots of a 20 – 40 ␮M solution of native or Arg91-modified cyt c were added, and the quenching of pyrene fluorescence by the heme of cyt c was observed. Substitution of Zn2ϩ for Fe2ϩ in the porphyrin of cyt c yields an intensely fluorescent derivative of cyt c [37] This analog has been characterized in considerable detail and has been shown to resemble closely the parent protein in most qualities representing a good model to study the conformation of cyt c [38]. The emission decays for [Zn2ϩ-heme]cyt c could be best fitted to a two-exponential process, yielding short (␶1) and long (␶2) lifetime components (Table I). This method gave the nucleotide/cyt c stoichiometry of 0.37 for the analog and 1.14 for the native protein. [Nle91]cyt c represents a model for the parent protein in which the only function compromised is ATP binding

RESULTS

DISCUSSION

Base leg Native cyt c