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Abstract: Adenylate kinase (AK) mediates the reversible transfer of phosphate groups between the adenylate nucleotides and contributes to the maintenance of their constant cellular level, necessary for energy metabolism and nucleic acid synthesis. The AK were purified from crude extracts of two sulfate-reducing bacteria (SRB), Desulfovibrio (D.) gigas NCIB 9332 and Desulfovibrio desulfuricans ATCC 27774, and biochemically and spectroscopically characterised in the native and fully cobalt- or zinc-substituted forms. These are the first reported adenylate kinases that bind either zinc or cobalt and are related to the subgroup of metal-containing AK found, in most cases, in Gram-positive bacteria. The electronic absorption spectrum is consistent with tetrahedral coordinated cobalt, predominantly via sulfur ligands, and is supported by EPR. The involvement of three cysteines in cobalt or zinc coordination was confirmed by chemical methods. Extended X-ray absorption fine structure (EXAFS) indicate that cobalt or zinc are bound by three cysteine residues and one histidine in the metal-binding site of the “LID” domain. The sequence 129Cys-X5-His-X15-Cys-X2-Cys of the AK from D. gigas is involved in metal coordination and represents a new type of binding motif that differs from other known zinc-binding sites of AK. Cobalt and zinc play a structural role in stabilizing the LID domain. [Copyright &y& Elsevier]

The thermodynamics of Cu(II) to Cu(I) reduction and thekineticsof the electron transfer (ET) process for Rhus verniciferastellacyanin (STC) immobilized on a decane-1-thiol coated gold electrodehave been measured through cyclic voltammetry at varying pH and temperature,in the presence of urea and in D2O. Immobilized STC undergoesa limited conformational change that mainly results in an enhancedexposure of one or both copper binding histidines to solvent whichslightly stabilizes the cupric state and increases histidine basicity.The large immobilization-induced increase in the pKafor the acid transition (from 4.5 to 6.3) makes thiselectrode–SAM–protein construct an attractive candidateas a biomolecular ET switch operating near neutral pH in molecularelectronics. Such a potential interest is increased by the robustnessof this interface against chemical unfolding as it undergoes onlymoderate changes in the reduction thermodynamics and in the ET ratein the presence of up to 8 M urea. The sensitivity of these parametersto solvent H/D isotope effects testifies to the role of protein solvationas effector of the thermodynamics and kinetics of ET. [ABSTRACT FROM AUTHOR]

A basic heme peroxidase has been isolated from cucumber (Cucumis sativus) peelings and characterized through electronic and 1H NMR spectra from pH 3 to 11. The protein, as isolated, contains a high-spin ferriheme which in the low pH region is sensitive to two acid–base equilibria with apparent pKa values of approximately 5 and 3.6, assigned to the distal histidine and to a heme propionate, respectively. At high pH, a new low-spin species develops with an apparent pKa of 11, likely due to the binding of an hydroxide ion to the sixth (axial) coordination position of the Fe(III). A number of acid–base equilibria involving heme propionates and residues in the distal cavity also affect the binding of inorganic anions such as cyanide, azide, and fluoride to the ferriheme, as well as the catalytic activity. The reduction potentials of the native protein and of its cyanide derivative, determined through UV–Vis spectroelectrochemistry, result to be −0.320 ± 0.015 and −0.412 ± 0.010 V, respectively. Overall, the reactivity of this protein parallels those of other plant peroxidases, especially horseradish peroxidase. However, some differences exist in the acid–base equilibria affecting its reactivity and in the reduction potential, likely as a result of small structural differences in the heme distal and proximal cavities. [Copyright &y& Elsevier]