Crystal structure of HydF scaffold protein provides insights into [FeFe]-hydrogenase maturation.

[FeFe]-hydrogenases catalyze the reversible production of H2 in some bacteria and unicellular eukaryotes. These enzymes require ancillary proteins to assemble the unique active site H-cluster, a complex structure composed of a 2Fe center bridged to a [4Fe-4S] cubane. The first crystal structure of a key factor in the maturation process, HydF, has been determined at 3 Å resolution. The protein monomer present in the asymmetric unit of the crystal comprises three domains: a GTP-binding domain, a dimerization domain, and a metal cluster-binding domain, all characterized by similar folding motifs. Two monomers dimerize, giving rise to a stable dimer, held together mainly by the formation of a continuous ß-sheet comprising eight ß-strands from two monomers. Moreover, in the structure presented, two dimers aggregate to form a supramolecular organization that represents an inactivated form of the HydF maturase. The crystal structure of the latter furnishes several clues about the events necessary for cluster generation/transfer and provides an excellent model to begin elucidating the structure/function of HydF in [FeFe]-hydrogenase maturation.

Stoichiometry of LHCI antenna polypeptides and characterization of gap and linker pigments in higher plants Photosystem I.

We report on the results obtained by measuring the stoichiometry of antenna polypeptides in Photosystem I (PSI) from Arabidopsis thaliana. This analysis was performed by quantification of Coomassie blue binding to individual LHCI polypeptides, fractionation by SDS/PAGE, and by the use of recombinant light harvesting complex of Photosystem I (Lhca) holoproteins as a standard reference. Our results show that a single copy of each Lhca1-4 polypeptide is present in Photosystem I. This is in agreement with the recent structural data on PSI-LHCI complex [Ben Shem, A., Frolow, F. and Nelson, N. (2003) Nature, 426, 630-635]. The discrepancy from earlier estimations based on pigment binding and yielding two copies of each LHCI polypeptide per PSI, is explained by the presence of 'gap' and 'linker' chlorophylls bound at the interface between PSI core and LHCI. We showed that these chlorophylls are lost when LHCI is detached from the PSI core moiety by detergent treatment and that gap and linker chlorophylls are both Chl a and Chl b. Carotenoid molecules are also found at this interface between LHCI and PSI core. Similar experiments, performed on PSII supercomplexes, showed that dissociation into individual pigment-proteins did not produce a significant loss of pigments, suggesting that gap and linker chlorophylls are a peculiar feature of Photosystem I.

cor Gene expression in barley mutants affected in chloroplast development and photosynthetic electron transport.

The expression of several barley (Hordeum vulgare) cold-regulated (cor) genes during cold acclimation was blocked in the albino mutant a(n), implying a chloroplast control on mRNAs accumulation. By using albino and xantha mutants ordered according to the step in chloroplast biogenesis affected, we show that the cold-dependent accumulation of cor14b, tmc-ap3, and blt14 mRNAs depends on plastid developmental stage. Plants acquire the ability to fully express cor genes only after the development of primary thylakoid membranes in their chloroplasts. To investigate the chloroplast-dependent mechanism involved in cor gene expression, the activity of a 643-bp cor14b promoter fragment was assayed in wild-type and albino mutant a(n) leaf explants using transient beta-glucuronidase reporter expression assay. Deletion analysis identified a 27-bp region between nucleotides -274 and -247 with respect to the transcription start point, encompassing a boundary of some element that contributes to the cold-induced expression of cor14b. However, cor14b promoter was equally active in green and in albino a(n) leaves, suggesting that chloroplast controls cor14b expression by posttranscriptional mechanisms. Barley mutants lacking either photosystem I or II reaction center complexes were then used to evaluate the effects of redox state of electron transport chain components on COR14b accumulation. In the mutants analyzed, the amount of COR14b protein, but not the steady-state level of the corresponding mRNA, was dependent on the redox state of the electron transport chain. Treatments of the vir-zb63 mutant with electron transport chain inhibitors showed that oxidized plastoquinone promotes COR14b accumulation, thus suggesting a molecular relationship between plastoquinone/plastoquinol pool and COR14b.