LAS TEORÍAS DE LA CULPABILIDAD DE LAS PERSONAS JURÍDICAS

PsaC is a relatively small protein, of about 9 kDa. It is highly conserved between different

I

species, and the differences that are present are conservative (Andersson and FranzérJ, 1992). 'It is coordinated by PsaD, as well as by PsaA and PsaB. PsaC shows a high degree of

sequence homology to soluble ferredoxins from bacteria, particularly to the Feptococcus aerogenes ferredoxin (Dunn and Gray, 1988; Golbeck, 1994). The only significant differences between the Peptococcus aerogenes ferredoxin and PsaC is that PsaC contains eight extra amino acids at the PsaC-terminal end of the protein, and an extra ten amino acids between the two iron-sulphur binding sites. They presumably enlarge the internal

loop structure as the polypeptide chain folds back on itself. Ferredoxins are soluble, whilst PsaC is a membrane bound protein. These extra amino acids may be responsible for the binding of PsaC to the P700-Fx core (Naver et a l, 1996). Deletion mutants of PsaC were created where the extra amino acids were deleted. The back reaction was less efficiently restored than in wild type cells, when PSI complexes were reconstituted with the modified PsaC. Moreover, the loop-deleted PsaC was unable to bind to the PSI core in the absence of PsaD (Naver et a l, 1996). These results show that the binding properties of PsaC were altered, and that the extra amino acids do have a role to play in the binding of PsaC.

PsaC coordinates the iron-sulphur centres F^ and Fg. These function as the tertiary electron acceptors, moving electrons out of the hydrophobic reaction centre core and into the hydrophilic stromal phase, further stabilising charge separation, and permitting soluble ferredoxin to be reduced with a high quantum efficiency (Yu et a l, 1993; Golbeck, 1994). The participation of F^ and Fg in electron transfer within PSI was demonstrated in cyanobacterial PSI complexes where PsaC, PsaD, and PsaE were removed. In these complexes, NADP^ photoreduction was lost. Photoreduction was observed again when PsaC and PsaD were rebound to the PSI core (Golbeck, 1994).

Fa and Fg are [4Fe-4S] iron-sulphur centres. These type of iron-sulphur centres are coordinated by four cysteine residues. In PsaC, nine conserved cysteine residues are present in the protein. Of these nine, eight are in the [4Fe-4S] iron-sulphur centre binding configuration CxxCxxCxxxCP. The Peptococcus aerogenes ferredoxin, which also binds 2 [4Fe-4S] centres, also has this [4Fe-4S] binding motif. In this protein, the first three cysteines of the motif provide three of the four ligands to the [4Fe-4S] iron-sulphur centre. The fourth cysteine of the motif provides the final ligand to the other [4Fe-4S] iron- sulphur centre. This geometry of the protein means that the two half chains do not form separate globular iron-sulphur domains. Each half chain cooperates in the same manner to form an approximately diad axis. The protein can be visualized as a dimer of two

connected polypeptides, containing shared [4Fe-4S] iron-sulphur clusters (Golbeck, 1994). PsaC probably exhibits similar folding patterns overall. However, the symmetry of the PsaC protein and the binding patterns of and Fg, means that the symmetry of the protein is imperfect. The two iron-sulphur centres therefore have slightly different spectroscopic and redox properties.

The pathway of electron transfer through PsaC is unknown. An option of linear and branched pathways exists. If the choice is made based solely on the equilibrium midpoints o f F^ (-530mV) and Fg (-580mV), a serial, linear pathway of electron flow is favoured, from Fx to Fg to F^. This is supported by the observation that, after selective chemical inactivation of Fg, photoreduction of F^ is inhibited at low temperatures (Malkin et al., 1984). However, evidence exists that indicates that electron transfer to F^ via Fg is not obligatory. It has been observed that when Fg is destroyed by HgCl2, an eight fold

decrease in the rate ofNADP^ photoreduction is seen (He and Malkin, 1994). Mutants have been created where one cysteine ligand for each iron-sulphur centre has been changed to an aspartate residue. In Anabaena variabilis ATCC 29413, the changes were Cl 3D (this cysteine binds Fg) and C50D (this cysteine binds F^) (Mannan et a l, 1996). In Synechocystsis, the changes were C14D (this cysteine binds Fg) and C51D (this cysteine binds Fa) (Zhao et a l, 1992). In both species, the changes resulted in the appropriate iron- sulphur centre becoming dysfunctional. The other iron-sulphur centre associated with PsaC functioned normally. It was found that F^ could be reduced in the presence o f a photochemically inactive Fg, and that no enhanced reduction of Fg was seen when F^ is rendered non functional. This data shows that Fg is not essential for electron transfer from PSI to ferredoxin. The question of the path of electron transfer through PsaC has therefore still to be resolved.

(II) The PSI Heterodimer

The core of PSI is composed of two proteins, PsaA and PsaB. These proteins form the catalytic core of PSI. They bind most of the 100 Chi a molecules that PSI possess, as well

as 12-15 p-carotene molecules (these have a role to play in light harvesting). PsaA and PsaB are both similar in size (83kDa) and are homologous to each other. Both proteins have been demonstrated to be light inducible in maize (Fish et a l, 1985). PsaA and PsaB are encoded by the chloroplast genes psaA and psaB. These two genes have been isolated and completely sequenced from maize (Fish et a l, 1985), tobacco (Shinozaki et a l, 1986), Liverwort (Ohyama et a l, 1986), and Chlamydomonas reinhardtii (Kück et a l, 1987), as well as several other plants and algae. In all cases, except Chlamydomonas reinhardtii, psaA and psaB are adjacent to one another, and cotranscribed. Analysis of their sequence shows that they are closely related, and the similarity between species is high: 95% similarity in higher plants, and 80% similarity between higher plants and cyanobacteria. PsaA and PsaB share an approximately 45% amino acid sequence similarity (Golbeck and Bryant, 1991; Fromme et a l, 1996; Chitnis et a l, 1995). The fact that these two proteins are so closely related appears to indicate that they probably arose from a gene duplication event of a single ancestral gene. They are believed to have evolved from the reaction centre protein of green sulphur bacteria and heliobacteriaia.

PsaA and PsaB are absolutely required for PSI assembly and function. PsaB has been inactivated in Synechocystis PCC.6803. PSI was completely disrupted in the mutant, while no effect was observed on PSII. While the expression of psaA was not found to be effected, the PsaA protein was not detected (Smart and McIntosh, 1993). Similarly, when expression of psaA was disrupted, PSI failed to assemble (Smart et a l, 1991).

Examination of the amino acid sequence shows the presence of short, hydrophobic regions, 19-25 residues long (Chitnis et a l, 1995; Cui et a l, 1994). These hydrophobic regions have the ability to integrate with the thylakoid membrane. Both PsaA and PsaB are both speculated to have 11 transmembrane helices, connected by hydrophilic loops. Thej 4 .5A

model of PSI from Synechococcus elongatus (Schubert et a l, 1996) predicts 29 membrane spanning helices for PSI. Of these, 22 are symmetrically related, and thought to arise from PsaA and PsaB. The majority of the charged residues of the two proteins are

found in the predicted extramembrane loops. These charged residues may be involved in interactions with smaller subunits, and diffusible electron carriers (Chitnis et a l, 1995). A large number of histidine residues are also found in the transmembrane helices, and are proposed to be involved in the coordination of Chi molecules (Fish et a l, 1985). A large number of conserved aspartate and glutamine residues are also present, and are likely to be involved in the binding of antenna Chi a molecules.

An interesting feature is seen in the amino acid sequence surrounding helices VIII and IX of both PsaA and PsaB. A ‘leucine zipper’ motif, similar to those found in DNA- binding proteins, is found in this region, where leucine residues are repeated at every eight (at least) residues. This arrangement of amino acids results in the protrusion o f the leucine residues out of the helix. Because this arrangement is found in both PsaA (four conserved leucines) and PsaB (five conserved leucines), the protruding leucines are able to act as a ‘zip’ (Kossel et a l, 1990). In PSI, it is likely that the mechanism exists to dimerize PsaA and PsaB, both of which are highly hydrophobic membrane proteins (Golbeck, 1994). However, conservative changes in PsaB of two of the leucine residues (L522V and L536M) involved in the zipper formation in Synechocystis PCC6803 had no effect on the growth characteristics of the organism or light induced charge separation of P700 to F^/Fg (Smart et a l, 1993).

Perhaps the most important and interesting region found in PsaA and PsaB is the extra stromal region connecting helices VIII and IX. This region contains a set of amino acids that are absolutely conserved in every species examined so far, and is thought to be involved in the binding of Fx..