Higher-order plants and mammals use similar mechanisms to repair and tolerate oxidative DNA damage. Most studies on the DNA repair process have focused on yeast and mammals, in which histone chaperone-mediated nucleosome disassembly/reassembly is essential for DNA to be accessible to repair machinery. However, little is known about the specific role and modulation of histone chaperones in the context of DNA damage in plants. Here, the histone chaperone NRP1, which is closely related to human SET/TAF-Iβ, was found to exhibit nucleosome assembly activity in vitro and to accumulate in the chromatin of Arabidopsis thaliana after DNA breaks. In addition, this work establishes that NRP1 binds to cytochrome c, thereby preventing the former from binding to histones. Since NRP1 interacts with cytochrome c at its earmuff domain, that is, its histone-binding domain, cytochrome c thus competes with core histones and hampers the activity of NRP1 as a histone chaperone. Altogether, the results obtained indicate that the underlying molecular mechanisms in nucleosome disassembly/reassembly are highly conserved throughout evolution, as inferred from the similar inhibition of plant NRP1 and human SET/TAF-Iβ by cytochrome c during DNA damage response.

Reaction centers of photosystem I contain three different [4Fe-4S] clusters named FX, FA, and FB. The terminal photosystem I acceptors (FA, FB) are distributed asymmetrically along the membrane normal, with one of them (FA or FB) being reduced from FX and the other one (FB or FA) reducing soluble ferredoxin. In the present work, kinetics of electron transfer has been measured in PSI from the cyanobacterium Synechocystis sp. PCC 6803 after inactivation of FB by treatment with HgCl2. Photovoltage measurements indicate that, in the absence of FB, reduction of FA by FX is still faster than the rate of FX reduction [(210 ns)-1]. Flash-absorption measurements show that the affinity of ferredoxin for HgCl2-treated PSI is only decreased by a factor of 3-4 compared to untreated photosystem I. The first-order rate of ferredoxin reduction by FA-, within the photosystem I/ferredoxin complex, has been calculated from measurements of P700+ decay. Compared to control PSI, this rate is several orders of magnitude smaller (6 s-1 versus 10(4)-10(6) s-1). Moreover, it is smaller than the rate of recombination from FA-, resulting in inefficient ferredoxin reduction (yield of 25%). After reconstitution of FB, about half of the reconstituted photosystem I reaction centers recover fast reduction of ferredoxin with kinetics similar to that of untreated photosystem I. These results support FB as the direct partner of ferredoxin and as the more distal cluster of photosystem I with respect to the thylakoid membrane, in accordance with a linear electron-transfer pathway FX-->FA-->FB-->ferredoxin.

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Programmed cell death is an event displayed by many different organisms along the evolutionary scale. In plants, programmed cell death is necessary for development and the hypersensitive response to stress or pathogenic infection. A common feature in programmed cell death across organisms is the translocation of cytochrome c from mitochondria to the cytosol. To better understand the role of cytochrome c in the onset of programmed cell death in plants, a proteomic approach was developed based on affinity chromatography and using Arabidopsis thaliana cytochrome c as bait. Using this approach, ten putative new cytochrome c partners were identified. Of these putative partners and as indicated by bimolecular fluorescence complementation, nine of them bind the heme protein in plant protoplasts and human cells as a heterologous system. The in vitro interaction between cytochrome c and such soluble cytochrome ctargets was further corroborated using surface plasmon resonance. Taken together, the results obtained in the study indicate that Arabidopsis thaliana cytochrome c interacts with several distinct proteins involved in protein folding, translational regulation, cell death, oxidative stress, DNA damage, energetic metabolism, and mRNA metabolism. Interestingly, some of these novel Arabidopsis thaliana cytochrome c-targets are closely related to those for Homo sapiens cytochrome c (Martínez-Fá bregas et al., unpublished). These results indicate that the evolutionarily well-conserved cytosolic cytochrome c, appearing in organisms from plants to mammals, interacts with a wide range of targets on programmed cell death.

Photosystem I reduction by the soluble metalloproteins cytochrome c(6) and plastocyanin, which are alternatively synthesized by some photosynthetic organisms depending on the relative availability of copper and iron, has been investigated in cyanobacteria, green algae and plants. The reaction mechanism is classified in three different types on the basis of the affinity of the membrane complex towards its electron donor protein. The role of electrostatic interactions in forming an intermediate transient complex, as well as the structural and functional similarities of cytochrome c(6) and plastocyanin are analysed from an evolutionary point of view. The proposal made is that the heme protein was first "discovered" by nature, when iron was much more abundant on the Earth's surface, and replaced by plastocyanin when copper became available because of the oxidizing conditions of the new atmosphere.

This paper reports the first site-directed mutagenesis analysis of any cytochrome c 6 , a heme protein that performs the same function as the copper-protein plastocyanin in the electron transport chain of photosynthetic organisms. Photosystem I reduction by the mutants of cytochrome c 6 from the cyanobacterium Synechocystis sp. PCC 6803 has been studied by laser flash absorption spectroscopy. Their kinetic efficiency and thermodynamic properties have been compared with those of plastocyanin mutants from the same organism. Such a comparative study reveals that aspartates at positions 70 and 72 in cytochrome c 6 are located in an acidic patch that may be isofunctional with the well known "southeast" patch of plastocyanin. Calculations of surface electrostatic potential distribution in the mutants of cytochrome c 6 and plastocyanin indicate that the changes in protein reactivity depend on the surface electrostatic potential pattern rather than on the net charge modification induced by mutagenesis. Phe-64, which is close to the heme group and may be the counterpart of Tyr-83 in plastocyanin, does not appear to be involved in the electron transfer to photosystem I. In contrast, Arg-67, which is at the edge of the cytochrome c 6 acidic area, seems to be crucial for the interaction with the reaction center.Cytochrome c 6 (Cyt) 1 and plastocyanin (Pc) are soluble metalloproteins, located inside the thylakoid lumen of photosynthetic organisms, that carry electrons from cytochrome b 6 f to photosystem I (PSI), which are both membrane-anchored complexes. Although cyanobacteria and eukaryotic green alga can synthesize both Pc and Cyt, Pc seems to have been able to replace the primitive Cyt along evolution of photosynthetic organisms as the copper-protein is the only electron carrier in higher plants. The two metalloproteins are now well characterized, both at the structural and functional levels. Their threedimensional structures have been solved by x-ray crystallography and NMR spectroscopy in several organisms, and their reaction mechanisms have been widely investigated (see Ref. 1 for a recent review).Alignment of eukaryotic Pc and Cyt molecules according to their dipole moment and surface charge distribution has allowed us the observation of areas in the heme protein similar to those previously reported in Pc: i) a hydrophobic region around the surface-exposed heme edge that could be equivalent to the north patch involving the copper ligand His-87 in Pc, and ii) a negatively charged area in Cyt similar to the east acidic patch around Tyr-83 in Pc (1, 2). It should be noted that such an acidic patch is significantly smaller in the metalloproteins isolated from prokaryotes, namely the cyanobacterium Synechocystis sp. PCC 6803 in which the acidic patch is rather southeast-facing, just below Tyr-87.In eukaryotic systems, site-directed mutagenesis of Pc has supplied relevant information on the role of specific residues in site 1 (or the hydrophobic north pole) and site 2 (or the acidic east face), which both have been propos...

Phosphorylation of tyrosine 48 of cytochrome c is related to a wide range of human diseases due to the pleiotropic role of the heme-protein in cell life and death. However, the structural conformation and physicochemical properties of phosphorylated cytochrome c are difficult to study as its yield from cell extracts is very low and its kinase remains unknown. Herein, we report a high-yielding synthesis of a close mimic of phosphorylated cytochrome c, developed by optimization of the synthesis of the non-canonical amino acid p-carboxymethyl-L-phenylalanine (pCMF) and its efficient site-specific incorporation at position 48. It is noteworthy that the Y48pCMF mutation significantly destabilizes the Fe-Met bond in the ferric form of cytochrome c, thereby lowering the pKa value for the alkaline transition of the heme-protein. This finding reveals the differential ability of the phosphomimic protein to drive certain events. This modified cytochrome c might be an important tool to investigate the role of the natural protein following phosphorylation.

A number of surface residues of cytochrome c 6 from the cyanobacterium Anabaena sp. PCC 7119 have been modified by site-directed mutagenesis. Changes were made in six amino acids, two near the heme group (Val-25 and Lys-29) and four in the positively charged patch (Lys-62, Arg-64, Lys-66, and Asp-72). The reactivity of mutants toward the membrane-anchored complex photosystem I was analyzed by laser flash absorption spectroscopy. The experimental results indicate that cytochrome c 6 possesses two areas involved in the redox interaction with photosystem I: 1) a positively charged patch that may drive its electrostatic attractive movement toward photosystem I to form a transient complex and 2) a hydrophobic region at the edge of the heme pocket that may provide the contact surface for the transfer of electrons to P 700 . The isofunctionality of these two areas with those found in plastocyanin (which acts as an alternative electron carrier playing the same role as cytochrome c 6 ) are evident.