Owing to its high thermal stability and structural simplicity, the archaebacterium
ThermoplasmaAcidophilum 20S proteasome was selected for mechanistic studies in this work. This oligomeric enzymecomplex consists of a barrel-shaped 20S core (~700kDa) comprised of four stacked seven-memberedrings with a
7777 subunit structure situated around a 7-fold symmetry axis. The hollow interior ofthe proteasome has three large interconnected chambers with narrow (13 Å diameter) entrances fromsolution located at either end of the barrel. The 14
-subunit proteolytic sites are located on the innersurface of the central chamber. Herein, we demonstrate that unfolded horse heart ferricytochrome
c (Cyt
c) is a novel chromophoric probe for investigation of the mechanism of proteasome action. Under conditionsof temperature and denaturant which unfold Cyt
c but do not alter the thermophilic proteasome, Cyt
c isextensively cleaved by the proteasome. Ten peptides were isolated and sequenced from the proteasomedigest. Analysis of the cleavage products established that unfolded Cyt
c and its covalently attached hemeprosthetic group are translocated to the central chamber where proteolysis occurs. In the presence ofsite-specific inhibitors of the proteasome, we demonstrate that unfolded cytochrome
c can be sequesteredinside the proteasome complex. Upon cooling, a quasistable host-guest complex is formed. Analysis ofthe complex via UV/visible spectroscopy and mass spectrometry gave evidence that the sequestered Cyt
c is essentially intact within the inhibited proteasome. High-performance liquid chromatography data showthat (1) complexes with an apparent stoichiometry of approximately one Cyt
c per proteasome can beformed and (2) when inhibition is removed from the complex, a rapid turnover of the sequestered Cyt
coccurs.