The reaction mechanism of electron transfer from the interchangeable metalloproteins plastocyanin (Pc) and cytochrome c6 (Cyt) to photooxidized P700 in photosystem I (PSI) has been studied by laser-flash absorption spectroscopy using a number of evolutionarily differentiated organisms such as cyanobacteria (Anabaena sp. PCC 7119 and Synechocystis sp. PCC 6803), green algae (Monoraphidium braunii), and higher plants (spinach). PSI reduction by Pc or Cyt shows different kinetics depending on the organism from which the photosystem and metalloproteins are isolated. According to the experimental data herein reported, three different kinetic models are proposed by assuming either an oriented collisional reaction mechanism (type I), a minimal two-step mechanism involving complex formation followed by intracomplex electron transfer (type II), or rearrangement of the reaction partners within the complex before electron transfer takes place (type III). Our findings suggest that PSI was able to first optimize its interaction with positively charged Cyt and that the evolutionary replacement of the ancestral Cyt by Pc, as well as the appearance of the fast kinetic phase in the Pc/PSI system of higher plants, would involve structural modifications in both the donor protein and PSI.

Since the first description of apoptosis four decades ago, great efforts have been made to elucidate, both in vivo and in vitro, the molecular mechanisms involved in its regulation. Although the role of cytochrome c during apoptosis is well established, relatively little is known about its participation in signaling pathways in vivo due to its essential role during respiration. To obtain a better understanding of the role of cytochrome c in the onset of apoptosis, we used a proteomic approach based on affinity chromatography with cytochrome c as bait in this study. In this approach, novel cytochrome c interaction partners were identified whose in vivo interaction and cellular localization were facilitated through bimolecular fluorescence complementation. Modeling of the complex interface between cytochrome c and its counterparts indicated the involvement of the surface surrounding the heme crevice of cytochrome c, in agreement with the vast majority of known redox adducts of cytochrome c. However, in contrast to the high turnover rate of the mitochondrial cytochrome c redox adducts, those occurring under apoptosis led to the formation of stable nucleo-cytoplasmic ensembles, as inferred mainly from surface plasmon resonance and nuclear magnetic resonance measurements, which permitted us to corroborate the formation of such complexes in vitro. The results obtained suggest that human cytochrome c interacts with pro-survival, anti-apoptotic proteins following its release into the cytoplasm. Thus, cytochrome c may interfere with cell survival pathways and unlock apoptosis in order to prevent the spatial and temporal coexistence of antagonist signals. Molecular & Cellular

The redox and acidlbase states and midpoint potentials of cytochrome b-559 have been determined in oxygenevolving photosystem I1 (PS 11) particles at room temperature in the pH range from 6.5 to 8.5. At pH 7.5 the fresh PS I1 particles present about 213 of their cytochrome b-559 in its reduced and protonated (non-autooxidizable) high-potential form and about 1/3 in its oxidized and non-protonated low-potential form. Potentiometric reductive titration shows that the protonated high-potential couple is pH-independent (Eo, + 380 mV), whereas the low-potential couple is non-protonated and pH-independent above pH 7.6 (Eo, pH > 7.6, + 140 mV), but becomes pH-dependent below this pH, with a slope of -72 mV/pH unit. Moreover, evidence is presented that in PS I1 particles cytochrome b-559 can cycle, according to its established redox and acidlbase properties, as an energy transducer at two alternate midpoint potentials and at two alternate pKa values. Red light absorbed by PS I1 induces reduction of cytochrome b-559 in these particles at room temperature, the reaction being completely blocked by dichlorophenyldimethylurea.The location and function of cytochrome b-559 in the chloroplast electron-transport chain(s), although widely and intensively investigated, have remained enigmatic and controversial [l -81. Understanding the oxidation/reduction potential and pH dependence of this cytochrome in high-potential (HP) low-potential (LP) forms is essential for assessing its role as both an electron carrier and a proton carrier, but in this regard the literature is ambiguous and includes incomplete and contradictory data. Equally important is deciding whether cytochrome b-559 is reduced or oxidized by light absorbed by photosystem 11, since the reported results are also contradictory in this respect. [15] this effect, which is reversible upon raising the pH, is inhibited by 3-(3',4'-dichloropheny1)-1,l'-dimethylurea (+ 395 mV at pH 7.8; + 335 mV at pH 5.0, and + 380 mV at pH 5.0 and in the presence of dichlorophenyldimethylurea). More recently our group [19 -211 obtained solid evidence that in spinach thylakoids the HP couple of cytochrome b-559 is pH-independent in the pH range between 6.5 and 8.5 (Eb, + 340 mV), whereas the LP couple is pH-independent above pH 7.6 (Eo, pH > 7.6, + 75 mV), but becomes pH-dependent below this pH, with a slope of -60 mV/pH unit. According to our proposal [6,21] the cytochrome functions at low potential as electron acceptor of PS 11, and at high potential as electron donor to PS I, thus acting as a transducer of redox energy into acidlbase energy between the two photosystems.The HP form of cytochrome b-559 is labile towards treatments, such as aging, sonication, mild heating, incubation in Tris buffer or Triton X-100, addition of either carbonylcyanide p-trifluoromethoxyphenylhydrazone or carbonylcyanide 3-chlorophenylhydrazone (CCCP), trypsin digestion, NaCl washing, that may alter or disrupt membrane structure and that cause what is usually considered an 'irreversible' conversion to the LP fo...