RESUMO
The influence of pi-interactions with a His ligand have been investigated in a family of copper-containing redox metalloproteins. The Met16Phe and Met16Trp pseudoazurin, and Leu12Phe spinach and Leu14Phe Phormidium laminosum plastocyanin variants possess active-site pi-contacts between the introduced residue and His81 and His87/92 respectively. The striking overlap of the side chain of Phe16 in the Met16Phe variant and that of Met16 in wild type pseudoazurin identifies that this position provides an important second coordination sphere interaction in both cases. His-ligand protonation and dissociation from Cu(I) occurs in the wild type proteins resulting in diminished redox activity, providing a [H(+)]-driven switch for regulating electron transfer. The introduced pi-interaction has opposing effects on the pKa for the His ligand in pseudoazurin and plastocyanin due to subtle differences in the pi-contact, stabilizing the coordinated form of pseudoazurin whereas in plastocyanin protonation and dissociation is favored. Replacement of Pro36, a residue that has been suggested to facilitate structural changes upon His ligand protonation, with a Gly, has little effect on the pKa of His87 in spinach plastocyanin. The mutations at Met16 have a significant influence on the reduction potential of pseudoazurin. Electron self-exchange is enhanced, whereas association with the physiological partner, nitrite reductase, is only affected by the Met16Phe mutation, but kcat is halved in both the Met16Phe and Met16Trp variants. Protonation of the His ligand is the feature most affected by the introduction of a pi-interaction.
Assuntos
Domínio Catalítico , Metaloproteínas/química , Metaloproteínas/metabolismo , Achromobacter cycloclastes/química , Achromobacter cycloclastes/genética , Achromobacter cycloclastes/metabolismo , Cobre/química , Cobre/metabolismo , Cristalografia por Raios X , Cianobactérias/química , Cianobactérias/genética , Cianobactérias/metabolismo , Dryopteris/química , Dryopteris/genética , Dryopteris/metabolismo , Elétrons , Concentração de Íons de Hidrogênio , Ligantes , Metaloproteínas/genética , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Oxirredução , Ligação Proteica , Estrutura Terciária de ProteínaRESUMO
The effect of pH on Cu(I) and Cu(II) forms of the isolated soluble domain of the stellacyanin from Rhus vernicifera (SCu), the Japanese lacquer tree, has been studied by electronic and NMR spectroscopy and using direct electrochemical measurements. A pK(a) value of 10.1-10.4 is observed for the alkaline transition in this oxidized phytocyanin and results in a slightly altered active-site structure, as indicated by changes in the visible and paramagnetic (1)H NMR spectra. Electrochemical studies show that the pK(a) value for this transition in SCu(I) (reduced SCu) is 11.0. These results are compared with those recently obtained for other phytocyanins, and in particular umecyanin. In all cases, the alkaline transition is caused by the deprotonation of the surface lysine residue adjacent to the axial ligand. This lysine residue is completely conserved in known phytocyanin sequences. Also highlighted in these studies are the remarkable active-site similarities between stellacyanin and umecyanin.
Assuntos
Metaloproteínas/química , Proteínas de Plantas/química , Sequência de Aminoácidos , Cobre , Concentração de Íons de Hidrogênio , Japão , Espectroscopia de Ressonância Magnética , Metaloproteínas/isolamento & purificação , Modelos Moleculares , Dados de Sequência Molecular , Proteínas de Plantas/isolamento & purificação , Conformação Proteica , Estrutura Secundária de Proteína , Rhus/química , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , EspectrofotometriaRESUMO
The effect of pH on the (1)H NMR spectrum, reduction potential, and self-exchange rate constant of the novel plastocyanin (PCu) from the fern plant Dryopteris crassirhizoma has been studied. The results are compared with those for the higher-plant PCu from parsley. In the (1)H NMR spectrum of D. crassirhizoma PCu(I), there is no sign that either of the His ligands is protonated at pH* down to 5.4. The reduction potentials of D. crassirhizoma and parsley PCu are 382 and 379 mV, respectively, at pH 7.4. When the pH value is decreased, the reduction potential of parsley PCu is seen to increase quite dramatically, consistent with protonation at His87 in PCu(I). A pK(a) of 5.8 is obtained from the electrochemistry data, consistent with a value of 5.6 determined by NMR. The reduction potential of D. crassirhizoma PCu exhibits a much less pronounced dependence on pH. The self-exchange rate constant of D. crassirhizomaPCu(I) is 3.4 x 10(3) M(-1) s(-1) at pH* 7.9. This is the smallest self-exchange rate constant reported to date for a PCu and can be rationalized by considering the altered distribution of charged residues on the surface of the D. crassirhizoma protein compared to the charge distributions of other higher-plant PCus. The self-exchange rate constant increases to 9 x 10(3) M(-1) s(-1) at pH* 5.4, consistent with enhanced protein-protein association at lower pH*, and the absence of His87 protonation in D. crassirhizoma PCu(I) in the accessible pH range.
