The iron-containing nitrile hydratase (NHase) is a photoreactive enzyme that is inactivated in the dark because of persistent association with NO and activated by photo-dissociation of NO. The crystal structure at 1.7 A resolution and mass spectrometry revealed the structure of the non-heme iron catalytic center in the nitrosylated state. Two Cys residues coordinated to the iron were post-translationally modified to Cys-sulfenic and -sulfinic acids. Together with another oxygen atom of the Ser ligand, these modifications induced a claw setting of oxygen atoms capturing an NO molecule. This unprecedented structure is likely to enable the photo-regulation of NHase and will provide an excellent model for designing photo-controllable chelate complexes and, ultimately, proteins.

Functional chaperone cooperation between Hsp70 (DnaK) and Hsp104 (ClpB) was demonstrated in vitro. In a eubacterium Thermus thermophilus, DnaK and DnaJ exist as a stable trigonal ring complex (TDnaK⅐J complex) and the dnaK gene cluster contains a clpB gene. When substrate proteins were heated at high temperature, none of the chaperones protected them from heat inactivation, but the TDnaK⅐J complex could suppress the aggregation of proteins in an ATP-and TGrpE-dependent manner. Subsequent incubation of these heated preparations at moderate temperature after addition of TClpB resulted in the efficient reactivation of the proteins. Reactivation was also observed, even though the yield was low, if the substrate protein alone was heated and incubated at moderate temperature with the TDnaK⅐J complex, TGrpE, TClpB, and ATP. Thus, all these components were necessary for the reactivation. Further, we found that TGroEL͞ES could not substitute TClpB.

Nitrile hydratase from Rhodococcus sp. N-771 is an ab heterodimer with a nonheme ferric iron in the catalytic center. In the catalytic center, aCys112 and aCys114 are modified to a cysteine sulfinic acid~Cys-SO 2 H! and a cysteine sulfenic acid~Cys-SOH!, respectively. To understand the function and the biogenic mechanism of these modified residues, we reconstituted the nitrile hydratase from recombinant unmodified subunits. The ab complex reconstituted under argon exhibited no activity. However, it gradually gained the enzymatic activity through aerobic incubation. ESI-LC0MS analysis showed that the anaerobically reconstituted ab complex did not have the modification of aCys112-SO 2 H and aerobic incubation induced the modification. The activity of the reconstituted ab complex correlated with the amount of aCys112-SO 2 H. Furthermore, ESI-LC0MS analyses of the tryptic digest of the reconstituted complex, removed of ferric iron at low pH and carboxamidomethylated without reduction, suggested that aCys114 is modified to Cys-SOH together with the sulfinic acid modification of aCys112. These results suggest that aCys112 and aCys114 are spontaneously oxidized to Cys-SO 2 H and Cys-SOH, respectively, and aCys112-SO 2 H is responsible for the catalytic activity solely or in combination with aCys114-SOH.

Various properties of semiconductor nanoparticles, including photoluminescence and catalytic activity, make these materials attractive for a range of applications. As nanoparticles readily coagulate and so lose their size-dependent properties, shape-persistent three-dimensional stabilizers that enfold nanoparticles have been exploited. However, such wrapping approaches also make the nanoparticles insensitive to external stimuli, and so may limit their application. The chaperonin proteins GroEL (from Escherichia coli) and T.th ('T.th cpn', from Thermus thermophilus HB8) encapsulate denatured proteins inside a cylindrical cavity; after refolding, the encapsulated proteins are released by the action of ATP inducing a conformational change of the cavity. Here we report that GroEL and T.th cpn can also enfold CdS semiconductor nanoparticles, giving them high thermal and chemical stability in aqueous media. Analogous to the biological function of the chaperonins, the nanoparticles can be readily released from the protein cavities by the action of ATP. We expect that integration of such biological mechanisms into materials science will open a door to conceptually new bioresponsive devices.