Biological catalysts (enzymes) speed up reactions by many orders of magnitude using fundamental physical processes to increase chemical reactivity. Hydrogen tunnelling has increasingly been found to contribute to enzyme reactions at room temperature. Tunnelling is the phenomenon by which a particle transfers through a reaction barrier as a result of its wave-like property. In reactions involving small molecules, the relative importance of tunnelling increases as the temperature is reduced. We have now investigated whether hydrogen tunnelling occurs at elevated temperatures in a biological system that functions physiologically under such conditions. Using a thermophilic alcohol dehydrogenase (ADH), we find that hydrogen tunnelling makes a significant contribution at 65 degrees C; this is analogous to previous findings with mesophilic ADH at 25 degrees C. Contrary to predictions for tunnelling through a rigid barrier, the tunnelling with the thermophilic ADH decreases at and below room temperature. These findings provide experimental evidence for a role of thermally excited enzyme fluctuations in modulating enzyme-catalysed bond cleavage.

We have isolated a chaperonin from the hyperthermophilic archaeon Sulfolobus solfataricus based on its ability to inhibit the spontaneous refolding at 50 "C of dimeric S. solfataricus malic enzyme. The chaperonin, a 920-kDa oligomer of 57-kDa subunits, displays a potassium-dependent ATPase activity with an optimum temperature at 80 "C. S. solfataricus chaperonin promotes correct refoldings of several guanidine hydrochloride-denatured enzymes from thermophilic and mesophilic sources. At a molar ratio of chaperonin oligomer to single polypeptide chain of 1: 1, S. solfataricus chaperonin completely inhibits spontaneous refoldings and suppresses aggregation upon dilution of the denaturant; refoldings resume upon ATP hydrolysis, with yields of active molecules and rates of folding notably higher than in spontaneous processes. S. solfataricus chaperonin prevents the irreversible inactivations at 90 "C of several thermophilic enzymes by the binding of the denaturation intermediate; the timecourses of inactivations are unaffected and most activity is regained upon hydrolysis of ATP. S. solfataricus chaperonin completely prevents the formation of aggregates during thermal inactivation of chicken egg white lysozyme at 70 "C, without affecting the rate of activity loss; ATP hydrolysis results in the recovery of most lytic activity. Tryptophan fluorescence measurements provide evidence that S. solfataricus chaperonin undergoes a dramatic conformational rearrangement in the presence of ATP/Mg, and that the hydrolysis of ATP is not required for the conformational change. The ATP/Mg-induced conformation of the chaperonin is fully unable to bind the protein substrates, probably due to disappearance or modification of the substrate binding sites. This is the first archaeal chaperonin whose involvement in protein folding has been demonstrated.

The Sulfolobus spindle virus, SSV2, encodes a tyrosine integrase which furthers provirus formation in host chromosomes. Consistently with the prediction made during sequence analysis, integration was found to occur in the downstream half of the tRNA(Gly) (CCC) gene. In this paper we report the findings of a comparative study of SSV2 physiology in the natural host, Sulfolobus islandicus REY15/4, versus the foreign host, Sulfolobus solfataricus, and provide evidence of differently regulated SSV2 life cycles in the two hosts. In fact, whereas a significant induction of SSV2 replication takes place during the growth of the natural host REY15/4, the cellular content of SSV2 DNA remains fairly low throughout the incubation of the foreign host. The accumulation of episomal DNA in the former case cannot be traced to decreased packaging activity because of a simultaneous increase in the virus titre in the medium. In addition, the interaction between SSV2 and its natural host is characterized by the concurrence of host growth inhibition and the induction of viral DNA replication. When this virus-host interaction was investigated using S. islandicus REY15A, a strain which is closely related to the natural host, it was found that the SSV2 replication process was induced in the same way as in the natural host REY15/4.

Protein disulfide oxidoreductases are ubiquitous redox enzymes that catalyse dithiol-disulfide exchange reactions with a CXXC sequence motif at their active site. A disulfide oxidoreductase, a highly thermostable protein, was isolated from Pyrococcus furiosus (PfPDO), which is characterized by two redox sites (CXXC) and an unusual molecular mass. Its 3D structure at high resolution suggests that it may be related to the multidomain protein disulfide-isomerase (PDI), which is currently known only in eukaryotes. This work focuses on the functional characterization of PfPDO as well as its relation to the eukaryotic PDIs. Assays of oxidative, reductive, and isomerase activities of PfPDO were performed, which revealed that the archaeal protein not only has oxidative and reductive activity, but also isomerase activity. On the basis of structural data, two single mutants (C35S and C146S) and a double mutant (C35S/C146S) of PfPDO were constructed and analyzed to elucidate the specific roles of the two redox sites. The results indicate that the CPYC site in the C-terminal half of the protein is fundamental to reductive/oxidative activity, whereas isomerase activity requires both active sites. In comparison with PDI, the ATPase activity was tested for PfPDO, which was found to be cation-dependent with a basic pH optimum and an optimum temperature of 90°C. These results and an investigation on genomic sequence databases indicate that PfPDO may be an ancestor of the eukaryotic PDI and belongs to a novel protein disulfide oxidoreductase family.Keywords: Archaea; protein disulfide-isomerase; protein disulfide oxidoreductase; Pyrococcus furiosus; redox sites.Protein disulfide oxidoreductases are ubiquitous redox enzymes that catalyse dithiol-disulfide exchange reactions. These enzymes share a CXXC sequence motif at their active sites. The two cysteines can undergo reversible oxidationreduction by shuttling between a dithiol and a disulfide form in the catalytic process. Protein disulfide oxidoreductases comprise the families of thioredoxin, glutaredoxin, protein disulfide-isomerase (PDI), and DsbA (disulfide-bond forming) and their homologs. Whereas thioredoxin and glutaredoxin mainly catalyse the reduction of disulfides, PDI and DsbA catalyse the formation or rearrangement of disulfide bridges in the protein-folding process.Protein disulfide oxidoreductases have been well studied in bacteria and eukarya, although to date only a few archaeal members of this protein family have been isolated, and therefore very little is known about protein disulfide oxidoreductases in archaea.A small redox protein with a molecular mass of 12 kDa was purified from the archaeon Methanobacterium thermoautotrophicum by McFarlan et al. [1]. This protein can catalyse the reduction of insulin disulfides and function as a hydrogen donor for Escherichia coli ribonucleotide reductase. The presence of the active-site motif CPYC, which is conserved in all glutaredoxins, suggested that it acts as a glutaredoxin-like protein. Surprisingly, however, the redu...

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