Recent structure determinations suggested a new binding site for a non-redox active metal ion in subunit I of cytochrome c oxidase both of mitochondrial and of bacterial origin. We analyzed the relevant metal composition of the bovine and the Paracoccus denitrificans enzyme and of bacterial sitedirected mutants in several residues presumably liganding this ion. Unlike the mitochondrial enzyme where a low, substoichiometric content of Ca 2+ was found, the bacterial wild-type (WT) oxidase showed a stoichiometry of one Ca per enzyme monomer. Mutants in Asp-477 (in immediate vicinity of this site) were clearly diminished in their Ca content and the isolated mutant enzyme revealed a spectral shift in the heme a visible absorption upon Ca addition, which was reversed by Na ions. This spectral behavior, largely comparable to that of the mitochondrial enzyme, was not observed for the bacterial WT oxidase. Further structure refinement revealed a tightly bound water molecule as an additional Ca 2+ ligand. z 1999 Federation of European Biochemical Societies.

The periplasmic Sud protein was previously isolated as a sulfide dehydrogenase from Wolinella succinogenes. Sud modified by a C-terminal His-tag (Sud-His 6 ) was produced in Escherichia coli by expression of the sud gene. Sud-His 6 catalyzed thiocyanate formation from cyanide and polysulfide. The V max of this activity was more than one order of magnitude higher than that of sulfide oxidation by dimethylnaphthoquinone and that of polysulfide reduction by BH Ϫ 4 . The apparent K m was less than 20 µM polysulfide. Polysulfide and not elemental sulfur was found to be the product of sulfide oxidation by dimethylnaphthoquinone, in contrast to the earlier view [Kreis-Kleinschmidt, V., Fahrenholz, F., Kojro, E. & Kröger, A. (1995) Arch. Microbiol. 165, 65Ϫ68]. Sud-His 6 did not contain metal ions or other prosthetic groups. Replacement by site-directed mutagenesis of the single cysteine residue of the Sud monomer caused complete loss of activity, while the exchange of the single histidine residue or of the lysine residue situated next to cysteine did not affect activity. In equilibrium dialysis, the Sud-His 6 monomer bound up to ten polysulfide sulfur atoms with a dissociation constant of 0.2 mM. Sud-His 6 loaded with polysulfide sulfur showed an absorption spectrum in the range of 350Ϫ400 nm ; this spectrum differed from that of free polysulfide. Electron transport from H 2 to polysulfide catalyzed by the membrane fraction of W. succinogenes was stimulated by the presence of small amounts of Sud-His 6. The apparent Km for polysulfide decreased sevenfold in the presence of saturating amounts of Sud-His 6 (1 µM Sud-His 6 dimer). Similar results were obtained with intact W. succinogenes cells containing low and high amounts of Sud. Sud appears to function as a polysulfide binding protein and probably binds polysulfide sulfur to its cysteine residue and transfers it to the substrate site of the membraneous polysulfide reductase.

Subunit II of the aa3 type cytochrome c oxidase contains a binuclear copper center (CuA) which functions as the entry point for electrons donated by cytochrome c. We have introduced site-specific mutations in residues liganding the CuA center in the oxidase of the bacterium Paracoccus denitrificans; the purified, fully assembled enzyme complexes were analyzed by various techniques, including EPR, optical spectroscopy, and total-reflection X-ray fluorescence spectrometry, to determine metal to protein ratios. In the C216S mutant, the binuclear CuA site is transformed into a mononuclear copper center. In contrast to wild type, the C216S mutant does no longer exhibit the characteristic absorption band in the near-infrared region of the optical spectrum that has been assigned to CuA. These major changes in the CuA site of this mutant correlate with an almost complete loss in catalytic activity.

Although the exact position of the substrate is not yet known, the residues lining the active site of Mch could be tentatively assigned. Comparison of Mch with the tetrahydrofolate-specific cyclohydrolase/dehydrogenase reveals similarities in domain arrangement and in some active-site residues, whereas the fold appears to be different. The adaptation of Mch to high salt concentrations and high temperatures is reflected by the excess of acidic residues at the trimer surface and by the higher oligomerization state of Mch compared with its mesophtic counterparts.

Sodium metal mirror reduction of biphenylene in aprotic THF solution containing [2.2.1]cryptand yields as a structurally characterized product the 9,9dimethylsilafluorene radical anion salt containing a (H 3 C) 2 Si expanded cyclobutadiene ring. In addition, the Si content can be detected by total reflection X-ray (TXRF) analysis. When the use of any silicon grease was avoided for all glass fittings, the solvent-separated biphenylene radical anion contact pair crystallized from an aprotic diglyme solution.