H-His-His-OH

Mono- and dinuclear Cu(II) complexes with Ac-PTVHNEYH-NH2 (L1) and Ac-NHHTLND-NH2 (L2) peptides from FomA protein of Fusobacterium nucleatum were studied by potentiometry, spectroscopic methods (UV-Vis, CD, EPR) and MS technique. The dominant mononuclear complexes for L1 ligand are: CuHL (pH range 5.0-6.0) with 2N {2Nim}, CuH-2L (pH range 8.0-8.5) and CuH-3L species (above pH 9.0) with 4N {Nim, 3N-} coordination modes. The complexes: CuH-1L with 3N {2Nim, N-}, CuH-2L with 3N {Nim, 2N-} and CuH-3L with 4N {Nim, 3N-} binding sites are proposed for the L2 ligand. Probably in the CuH-2L complex for CuL2 system the second His residue in His-His sequence is bound to Cu(II) ion, while the first His residue may stabilize this complex by His-His and/or His-Cu(II) interactions. The dominant dinuclear Cu2L1 complexes in the pH range 6.5-10.5 are: the Cu2H-4L and Cu2H-6L species with 3N{Nim, 2N-}4N{Nim, 3N-} and 4N{Nim, 3N-}4N{Nim, 3N-} binding sites, respectively. In the case of the Cu2L2 complex in the pH range 7.2-10.5, the Cu2H-4L and Cu2H-7L species dominate with 2N{Nim, N-}4N{Nim, 3N-} and (Cu(OH)42-4N{Nim, 3N-}) coordination modes, respectively. The ability to generate reactive oxygen species (ROS) by uncomplexed Cu(II) ions, ligands and their complexes at pH 7.4 in the presence of hydrogen peroxide or ascorbic acid was studied. UV-Vis, luminescence, EPR spin trapping and gel electrophoresis methods were used. Both complexes produce higher level of ROS compared to those of their ligands. ROS produced by Cu(II) complexes are hydroxyl radical and singlet oxygen, which contribute to oxidative DNA cleavage.

The solution chemical properties, superoxide dismutase and catecholase activity of the copper(ii)-Ac-His-His-Gly-His-OH (hhgh) complexes were studied to identify functional and structural models of copper-containing oxidases. The solution speciation was determined in the pH range 3-11 by two independent methods (potentiometry and pH-dependent EPR measurements). The results obtained by the two methods agree very well with each other and show the formation of differently protonated CuH(x)L complexes (where x= 2 ,1, 0, -1, -2, -3) in aqueous solution. The spectroscopic (UV-Vis, CD, EPR) data indicate that the coordination of the imidazole rings is a determinant factor in all these complexes. Amide coordinated complexes are dominant only above pH 8. This offers excellent possibilities for structural/functional modelling of copper(ii) containing metalloenzymes. Indeed, the {3N(im)} coordinated CuL species (pH = 6-7) has efficient superoxide dismutase-like activity. The {3N(im),OH(-)} coordinated CuH(-1)L possesses outstanding activity to catalyze the oxidation of 3,5-di-tert-butylcatechol (H(2)dtbc) by dioxygen in 86 wt% methanol-water, providing the first example that copper(ii)-peptide complexes are able to mimic copper containing oxidases.

The mitochondrial carnitine/acylcarnitine carrier (CAC) of Rattus norvegicus contains two His, His-29 and His-205. Only the first residue is conserved in all the members of the CAC subfamily and is positioned before the first of the three conserved motifs. In the homology model of CAC, His-29 is located in H1 close to the bottom of the central cavity. His-205 is the first amino acid of H5 and it is exposed towards the cytosol. The effect of substitution of the His residues on the transport function of the reconstituted mutant CACs has been analysed, in comparison with the wild-type. H29A showed very low activity, H29K and H29D were nearly inactive, whereas H205A, H205K and H205D showed activities similar to that of the wild-type. His-29 has also been substituted with Gln, Asn, Phe and Tyr. All the mutants showed very low transport function and, similarly to H29A, higher Km, reduced Vmax and altered selectivity towards (n)acylcarnitines, with the exception of H29Q, which exhibited functional properties similar to those of the wild-type. The experimental data, together with a comparative analysis of the carnitine acyltranferase active sites, indicated that His-29 forms an H-bond with the beta-OH of carnitine. The substitution of His-205 led to a change of response of the CAC to the pH. The results are discussed in terms of relationships of His-29 with the molecular mechanism of translocation of the CAC.