The formation of two metal-thiolate clusters in rabbit liver metallothionein 2 (MT) has been examined by 113Cd NMR spectroscopy at pH 7.2 and 8.6. The chemical shifts of the 113Cd resonances developing in the course of apoMT titration with 113Cd(II) ions have been compared with those of fully metal occupied 113Cd7-MT. At pH 7.2 and at low metal occupancy (less than 4), a cooperative formation of the four-metal cluster (cluster A) occurs. Further addition of 113Cd(II) ions generates all the resonances of the three-metal cluster (cluster B) in succession, suggesting cooperative metal binding to this cluster also. In contrast, similar studies at pH 8.6, at low metal occupancy (less than 4), reveal a broad NMR signal centered at 688 ppm. This observation indicates that an entirely different protein structure exists. When exactly 4 equiv of 113Cd(II) are bound to apoMT, the 113Cd NMR spectrum changes to the characteristic spectrum of cluster A. Further addition of 113Cd(II) ions again leads to the cooperative formation of cluster B. These results stress the determining role of the cluster A domain on the overall protein fold. The observed pH dependence of the cluster formation in MT can be rationalized by the different degree of deprotonation of the cysteine residues (pKa approximately 8.9), i.e., by the difference in the Gibbs free energy required to bind Cd(II) ions to the thiolate ligands at both pH values.

Carbon-13 and proton NMR. spectra o€ pentafulvene and of a series of 6-substituted fulvenes have been analysed and assigned by homo-and heteronuclcar double resonance and with the aid of iterative computation. 13C and l H chemical shifts are interpreted in tcrms of substituent effects and compared with z-electron charges calculated for the unsubstituted fulvene. From I3C shifts a 10 percent contribution of dipolar structures to the electronic configuration of iulvene may be estimated. ,411 long-range proton-proton coupling constants including relative signs and some proton-carbon couplings in the fulvene spin system have been determined and assigned. l)

In mammalian metallothioneins the metals are organized in two adamantane-type clusters with three and four metal ions which are tetrahedrally coordinated by thiolate ligands. The metal selectivity of the metal-thiolate clusters in rabbit liver metallothionein has been studied by offering two ions, i. e. Co(II)/Cd(II), Zn(II)/Cd(II) or Co(II)/Zn(II), to the metal-free protein. The heterogeneous metal complexes thus formed were characterized by electronic absorption, magnetic circular dichroism, 'I3Cd-NMR and EPR spectroscopy. In the case of Co/Cdmetallothionein, homometallic cluster occupation occurs, with the Cd(I1) ions bound exclusively to the four-metal cluster. In contrast, heterometallic clusters were formed for both Zn/Cd-and Co/Zn-metallothionein. Based on evidence from corresponding inorganic structures of adamantane metal-thiolate cages, it is suggested that the major factor governing the cluster type is the protein structure perturbation due to the cluster volume variations. Thus, while metal thiolate affinities are important in the folding process, size-match selectivity is the dominant factor in the metal-loaded protein.Metallothioneins are members of a class of proteins, ubiquitous in nature, which are characterized by their unusual amino acid composition and structure. These proteins are rich in cysteine residues and bind with a high affinity d" metal ions such as Zn(II), Cd(II), Cu(1) and Hg(I1). Since the biosynthesis of metallothioneins is induced by these metals and by certain hormones, it is believed that they play a crucial role in the physiological handling of the essential trace metals zinc and copper, and in the detoxification of the non-essential metals cadmium and mercury [l, 21. Mammalian metallothioneins contain 61 or 62 amino acids out of which 20 are conserved cysteine residues involved in the binding of 7 divalent metal ions, i.e. Zn and/or Cd [3]. From spectroscopic and chemical studies, the presence of two adamantane-like metal-thiolate clusters of three and four metal ions tetrahedrally coordinated by thiolate ligands has been suggested [3 -81. The determination of the three-dimensional structure of metallothionein in crystals, by X-ray diffraction 191, and in solution, by two-dimensional NMR [lo -141, confirmed the spectroscopic cluster model. However, both three-dimensional models showed marked differences regarding the assignment of the sequence-specific cysteine-metal coordination bonds. The correctness of the NMR model has been confirmed by the recent reinvestigation of the crystal structure [ 151.Several lines of evidence suggest that the two clusters in metallothionein exhibit differential affinities for Cd, with the tighter binding occurring in the four-metal cluster [5, 16 -181. interestingly, although the Zn,-metallothionein form has been isolated from mammalian tissue, the highest cadmium content in metallothionein isolated from Cd-exposed animals was 5 mol Cd/mol protein [l], the remainder being Zn [19]. Moreover, in the native Znz,Cd5-metallothionein form the two met...

The 1-deoxy-1-nitro-D-manno-pyranose 4 was transformed into the nitroolefin 5 and hence into the anomeric 1,2-dideoxyl-nitro-3,4,6-tri-O-benzyl-~-arabinohexopyranoses (3 a and 3b; c j the Scheme). Conformational analysis of 1 -benzyloxy-2-nitroethane (6) by 'H-NMR spectroscopy (Fig. 2) showed the synclinal conformation to be more stable than the antiperiplanar one by about 1.4 kcal/mol (attractive gauche-effect). This gauche-effect favours the 1-deoxyl-nitro-2,3,4,6-tetra-Obenzyl-B-D-manno-hexopyranose (1 b) possessing an equatorial nitro group, which is, however, qualitatively the less stable anomer. The relative concentrations of the anorners of 1 and 3, respectively, were determined by 'H-NMR spectroscopy after base catalyzed equilibration at 37" in CHC13-solution (Table). Anomeric effects for the nitro group of approximately 2.4 kcal/mol in 3 and of approximately 3.4 kcal/ mol in 1 were calculated.

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