The crystal structure of the ferritin from the archaeon, hyperthermophile and anaerobe Pyrococcus furiosus (PfFtn) is presented. While many ferritin structures from bacteria to mammals have been reported, until now only one was available from archaea, the ferritin from Archaeoglobus fulgidus (AfFtn). The PfFtn 24-mer exhibits the 432 point-group symmetry that is characteristic of most ferritins, which suggests that the 23 symmetry found in the previously reported AfFtn is not a common feature of archaeal ferritins. Consequently, the four large pores that were found in AfFtn are not present in PfFtn. The structure has been solved by molecular replacement and refined at 2.75-Å resolution to R = 0.195 and R free = 0.247. The ferroxidase center of the aerobically crystallized ferritin contains one iron at site A and shows sites B and C only upon iron or zinc soaking. Electron paramagnetic resonance studies suggest this iron depletion of the native ferroxidase center to be a result of a complexation of iron by the crystallization salt. The extreme thermostability of PfFtn is compared with that of eight structurally similar ferritins and is proposed to originate mostly from the observed high number of intrasubunit hydrogen bonds. A preservation of the monomer fold, rather than the 24-mer assembly, appears to be the most important factor that protects the ferritin from inactivation by heat.

A ferritin from the obligate anaerobe and hyperthermophilic archaeon Pyrococcus furiosus (optimal growth at 100 degrees C) has been cloned and overproduced in Escherichia coli to one-fourth of total cell-free extract protein, and has been purified in one step to homogeneity. The ferritin (PfFtn) is structurally similar to known bacterial and eukaryal ferritins; it is a 24-mer of 20 kDa subunits, which add up to a total Mr 480 kDa. The protein belongs to the non-heme type of ferritins. The 24-mer contains approximately 17 Fe (as isolated), 2,700 Fe (fully loaded), or <1 Fe (apoprotein). Fe-loaded protein exhibits an EPR spectrum characteristic for superparamagnetic core formation. At 25 degrees C V(max) = 25 micromole core Fe(3+) formed per min per mg protein when measured at 315 nm, and the K(0.5) = 5 mM Fe(II). At 0.3 mM Fe(II) activity increases 100-fold from 25 to 85 degrees C. The wild-type ferritin is detected in P. furiosus grown on starch. PfFtn is extremely thermostable; its activity has a half-life of 48 h at 100 degrees C and 85 min at 120 degrees C. No apparent melting temperature was found up to 120 degrees C. The extreme thermostability of PfFtn has potential value for biotechnological applications.

Recombinant ferritin from Pyrococcus furiosus expressed in Escherichia coli exhibits in EPR monitored redox titrations a mixed valence (Fe 3+ -Fe 2+ ) S = 1/2 signal at intermediate potentials that is a high-resolution homolog of the ferroxidase signal previously described for reconstituted horse spleen apo-ferritin. P. furiosus reconstituted apo-ferritin reduced to intermediate potentials exhibits the same mixed-valence signal, which integrates to close to one spin per subunit. The reduction potentials of +210 and +50 mV imply that the iron dimer is a stable prosthetic group with three redox states.

Crystals of the title protein have been produced and preliminary structural analysis has been carried out. The crystals belong to the orthorhombic space group C222 1 , with unit-cell parameters a = 258.1, b = 340.1, c = 266.5 Å . The protein forms a 24-mer of 20 kDa subunits, which assemble with 432 noncrystallographic symmetry. A total of 36 monomers are found in the asymmetric unit, corresponding to one and a half 24-mers.

The pyruvate dehydrogenase complex (PDC) plays a central role in cellular metabolism and regulation. As a metabolite-channeling multi-enzyme complex it acts as a complete nanomachine due to its unique geometry and by coupling a cascade of catalytic reactions using 'swinging arms'. Mammalian and specifically human PDC (hPDC) is assembled from multiple copies of E1 and E3 bound to a large E2/E3BP 60-meric core. A less restrictive and smaller catalytic core, which is still active, is highly desired for both fundamental research on channeling mechanisms and also to create a basis for further modification and engineering of new enzyme cascades. Here, we present the first experimental results of the successful disintegration of the E2/E3BP core while retaining its activity. This was achieved by C-terminal α-helixes double truncations (eight residues from E2 and seven residues from E3BP). Disintegration of the hPDC core via double truncations led to the formation of highly active (approximately 70% of wildtype) apparently unordered clusters or agglomerates and inactive non-agglomerated species (hexamer/trimer). After additional deletion of N-terminal 'swinging arms', the aforementioned C-terminal truncations also caused the formation of agglomerates of minimized E2/E3BP complexes. It is likely that these 'swinging arm' regions are not solely responsible for the formation of the large agglomerates.