Programmed death (apoptosis) is turned on in damaged or unwanted cells to secure their clean and safe self-elimination. The initial apoptotic events are coordinated in mitochondria, whereby several proapoptotic factors, including cytochrome c, are released into the cytosol to trigger caspase cascades. The release mechanisms include interactions of B-cell/lymphoma 2 family proteins with a mitochondria-specific phospholipid, cardiolipin, to cause permeabilization of the outer mitochondrial membrane. Using oxidative lipidomics, we showed that cardiolipin is the only phospholipid in mitochondria that undergoes early oxidation during apoptosis. The oxidation is catalyzed by a cardiolipin-specific peroxidase activity of cardiolipin-bound cytochrome c. In a previously undescribed step in apoptosis, we showed that oxidized cardiolipin is required for the release of proapoptotic factors. These results provide insight into the role of reactive oxygen species in triggering the cell-death pathway and describe an early role for cytochrome c before caspase activation.

During apoptosis, cytochrome c (cyt c) is released from intermembrane space of mitochondria into the cytosol where it triggers caspase-dependent machinery. We discovered that cyt c plays another critical role in early apoptosis as a cardiolipin (CL)-specific oxygenase to produce CL hydroperoxides required for release of pro-apoptotic factors. We quantitatively characterized the activation of peroxidase activity of cyt c by CL and hydrogen peroxide. At low ionic strength and high CL/cyt c ratios, peroxidase activity of CL/cyt c complex was increased >50 times. This catalytic activity correlated with partial unfolding of cyt c monitored by Trp 59 fluorescence and absorbance at 695 nm (Fe-S(Met 80) band). The peroxidase activity increase preceded the loss of protein tertiary structure. Monounsaturated tetraoleoyl-CL (TOCL) induced peroxidase activity and unfolding of cyt c more effectively than saturated tetramyristoyl-CL (TMCL). TOCL/cyt c complex was found more resistant to dissociation by high salt concentration. These findings suggest that electrostatic CL/cyt c interactions are central to the initiation of the peroxidase activity, while hydrophobic interactions are involved when cyt c's tertiary structure is lost. In the presence of CL, cyt c peroxidase activity is activated at lower H 2 O 2 concentrations than for isolated cyt c molecules. This suggests that redistribution of CL in the mitochondrial membranes combined with increased production of H 2 O 2 can switch on the peroxidase activity of cyt c and CL oxidation in mitochondria-a required step in execution of apoptosis.

Upon interaction with anionic phospholipids, particularly mitochondria-specific cardiolipin (CL), cytochrome c (cyt c) loses its tertiary structure and its peroxidase activity dramatically increases. CL induced peroxidase activity of cyt c has been found to be important for selective CL oxidation in cells undergoing programmed death. During apoptosis, the peroxidase activity and the fraction of CL-bound cyt c markedly increases suggesting that CL may act as a switch to regulate cyt c's mitochondrial functions. Using cyclic voltammetry and equilibrium redox-titrations, we show that the redox potential of cyt c shifts negatively by 350-400 mV upon binding to CL-containing membranes. Consequently, functions of cyt c as an electron transporter and cyt c reduction by Complex III are strongly inhibited. Further, CL/cyt c complexes are not effective in scavenging superoxide anions and are not effectively reduced by ascorbate. Thus, both redox properties and functions of cyt c change upon interaction with CL in the mitochondrial membrane, diminishing cyt c's electron donor/acceptor role and stimulating its peroxidase activity.

Quantum mechanical analysis of electron tunneling in nine thermally fluctuating cytochrome b562 derivatives reveals two distinct protein-mediated coupling limits. A structure-insensitive regime arises for redox partners coupled through dynamically averaged multiple-coupling pathways (in seven of the nine derivatives) where heme-edge coupling leads to the multiple-pathway regime. A structure-dependent limit governs redox partners coupled through a dominant pathway (in two of the nine derivatives) where axial-ligand coupling generates the single-pathway limit and slower rates. This two-regime paradigm provides a unified description of electron transfer rates in 26 ruthenium-modified heme and blue-copper proteins, as well as in numerous photosynthetic proteins.

Electron transfer (ET) processes in DNA are of current interest because of their involvement in oxidative strand cleavage reactions and their relevance to the development of molecular electronics. Two mechanisms have been identified for ET in DNA, a single-step tunneling process and a multistep charge-hopping process. The dynamics of tunneling reactions depend on both the distance between the electron donor and acceptor and the nature of the molecular bridge separating the donor and acceptor. In the case of protein and alkane bridges, the distance dependence is not strongly dependent on the properties of the donor and acceptor. In contrast, we show here that the distance decay of DNA ET rates varies markedly with the energetics of the donor and acceptor relative to the bridge. Specifically, we find that an increase in the energy of the bridge states by 0.25 eV (1 eV ‫؍‬ 1.602 ؋ 10 ؊19 J) relative to the donor and acceptor energies for photochemical oxidation of nucleotides, without changing the reaction free energy, results in an increase in the characteristic exponential distance decay constant for the ET rates from 0.71 to 1.1 Å ؊1 . These results show that, in the small tunneling energy gap regime of DNA ET, the distance dependence is not universal; it varies strongly with the tunneling energy gap. These DNA ET reactions fill a ''missing link'' or transition regime between the large barrier (rapidly decaying) tunneling regime and the (slowly decaying) hopping regime in the general theory of bridge-mediated ET processes. E lectron transfer (ET) processes in which an electron donor and acceptor are separated by a molecular spacer or bridge (D-B-A systems) are encountered widely in biological systems (proteins and DNA; refs. 1-4) and molecular wires (5-7). The dynamics of such processes are known to depend, inter alia, on the length and nature of the bridge (8, 9). The dynamics of single-step photoinduced ET in D-B-A systems, a process referred to as tunneling, is generally found to display an exponential dependence on the D-A distance as described by Eq. 1,where k o is a temperature-dependent prefactor, r da is the D-A separation, and ␤ characterizes the steepness of the experimental distance dependence are observed for bridges consisting of conjugated polyenes (5, 12), which probably arise from delocalization of the donor and acceptor states onto the bridge. DNA systems apparently are unique in that a wide range of values of ␤ (0.1-1.5 Å Ϫ1 ) have been observed for similar duplex DNA bridge structures (13,14). The very smallest ␤ values for DNA ET may arise from an alternative ET mechanism, multistep hole hopping (15-18). The distance dependence of the reorganization energy may enhance some of the observed ␤ values (19). A question in DNA ET is how much the donor and acceptor energetics can influence the ␤ value by tuning the electronic coupling strength. By making donor-acceptor modifications that do not change the ETactivation free energies, we find that ␤ values can be changed by more than 50%. We interpret...

pH-Induced conformational switching is essential for functioning of diphtheria toxin, which undergoes a membrane insertion/translocation transition triggered by endosomal acidification as a key step of cellular entry. In order to establish the sequence of molecular rearrangements and side chain protonation accompanying the formation of the membrane-competent state of the toxin’s translocation (T) domain, we have developed and applied an integrated approach that combines multiple techniques of computational chemistry (e.g., long, µsec-range, all-atom molecular dynamics simulations; continuum electrostatics calculations; and thermodynamic integration) with several experimental techniques of fluorescence spectroscopy. Thermodynamic integration calculations indicate that protonation of H257 causes the greatest destabilization of the native structure (6.9 kcal/mole), which is consistent with our early mutagenesis results. Extensive equilibrium MD simulations with a combined length of over eight µsec demonstrate that histidine protonation, while not accompanied by the loss of structural compactness of the T-domain, nevertheless results in substantial molecular rearrangements characterized by the partial loss of secondary structure due to unfolding of helices TH1 and TH2, and the loss of close contact between the C- and N-terminal segments. The structural changes accompanying the formation of the membrane-competent state ensure an easier exposure of the internal hydrophobic hairpin formed by helices TH8 and TH9, in preparation for its subsequent transmembrane insertion.

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