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The induction of apoptosis by p53 in response to cellular stress is its most conserved function and crucial for p53 tumor suppression. We recently reported that p53 directly induces oligomerization of the BH1,2,3 effector protein Bak, leading to outer mitochondrial membrane permeabilization (OMMP) with release of apoptotic activator proteins. One important mechanism by which p53 achieves OMMP is by forming an inhibitory complex with the antiapoptotic BclXL protein. In contrast, the p53 complex with the Bcl2 homolog has not been interrogated. Here we have undertaken a detailed characterization of the p53-Bcl2 interaction using structural, biophysical, and mutational analyses. We have identified the p53 DNA binding domain as the binding interface for Bcl2 using solution NMR. The affinity of the p53-Bcl2 complex was determined by surface plasmon resonance analysis (BIAcore) to have a dominant component K D 535 ؎ 24 nM. Moreover, in contrast to wild type p53, endogenous missense mutants of p53 are unable to form complexes with endogenous Bcl2 in human cancer cells. Functionally, these mutants are all completely or strongly compromised in mediating OMMP, as measured by cytochrome c release from isolated mitochondria. These data implicate p53-Bcl2 complexes in contributing to the direct mitochondrial p53 pathway of apoptosis and further support the notion that the DNA binding domain of p53 is a dual function domain, mediating both its transactivation function and its direct mitochondrial apoptotic function.A major function of the p53 tumor suppressor is the induction of an apoptotic program in response to a broad variety of cell stresses. Thus, understanding the mechanisms by which p53 executes cell death pathways is of considerable importance in cancer biology. The basis for the powerful apoptotic and tumor suppressor activity of p53 lies in its pleiotropism, which includes transcription-dependent and transcription-independent functions (1, 2). p53-mediated apoptosis primarily signals through the mitochondrial pathway (1). Some notable p53 target genes such as the BH3-only proteins PUMA, Noxa, Bax, and p53AIP1 reside and/or act at the mitochondria (3-7).We previously showed that in response to a death stimulus such as DNA damage or hypoxia, a fraction of stabilized p53 rapidly translocates to mitochondria in primary, immortal, and transformed cells (8 -11). The functional consequences of this phenomenon were revealed by targeting exogenous p53 to mitochondria in p53 null cells. Mitochondrially targeted p53 was sufficient to launch apoptosis and suppress colony formation directly from the mitochondrial platform in a transcription-independent fashion (9, 10). Translocated endogenous mitochondrial p53 interacts with anti-apoptotic BclXL and Bcl2 proteins and blocks their functions. Purified p53 protein induces oligomerization of Bak and permeabilization of the outer mitochondrial membrane and strongly promotes cytochrome c release from healthy unstressed mitochondria (10).Using computational and mutational analyses, we ...

Intermolecular electron transfer (ET) between the free phenothiazine donor (PH) and its cation radical (PH*+) proceeds via the [1:1] precursor complex (PH)(2)*+ which is transiently observed for the first time by its diagnostic (charge-resonance) absorption band in the near-IR region. Similar intervalence (optical) transitions are also observed in mixed-valence cation radicals with the generic representation: P(br)P*+, in which two phenothiazine redox centers are interlinked by p-phenylene, o-xylylene, and o-phenylene (br) bridges. Mulliken-Hush analysis of the intervalence (charge-resonance) bands afford reliable values of the electronic coupling element H(IV) based on the separation parameters for (P/P*+) centers estimated from some X-ray structures of the intermolecular (PH)(2)*+ and the intramolecular P(br)P*+ systems. The values of H(IV), together with the reorganization energies lambda derived from the intervalence transitions, yield activation barriers DeltaG(ET)() and first-order rate constants k(ET) for electron-transfer based on the Marcus-Hush (two-state) formalism. Such theoretically based values of the intrinsic barrier and ET rate constants agree with the experimental activation barrier (E(a)) and the self-exchange rate constant (k(SE)) independently determined by ESR line broadening measurements. This convergence validates the use of the two-state model to adequately evaluate the critical electronic coupling elements between (P/P*+) redox centers in both (a) intermolecular ET via the precursor complex and (b) intramolecular ET within bridged mixed-valence cation radicals. Important to intermolecular ET mechanism is the intervention of the strongly coupled precursor complex since it leads to electron-transfer rates of self-exchange that are 2 orders of magnitude faster (and activation barrier that is substantially lower) than otherwise predicted solely on the basis of Marcus reorganization energy.