Effect of histidine 6 protonation on the active site structure and electron-transfer capabilities of pseudoazurin from Achromobacter cycloclastes.
The paramagnetic (1)H NMR spectrum of Cu(II) pseudoazurin [PACu(II)] contains eight directly observed hyperfine-shifted resonances which we have assigned using saturation transfer experiments on a 1:1 mixture of PACu(I) and PACu(II). The spectrum exhibits a number of similarities to those of other cupredoxins, but differences are found concerning the Cu-S(Met) interaction. The spectrum is dependent on pH* in the range 8.5-4.5 (pK(a)* 6.4), and a conformational change involving movement of the copper ion away from the Met toward the equatorial ligands, as a consequence of protonation of the surface His6 residue, is identified. Corresponding changes are also seen in the UV/vis spectrum. The protonation/deprotonation equilibrium of His6 influences the reduction potential of the protein in the same pH range. The self-exchange rate constant of PACu at pH* 6.0 (25 degrees C) is considerably smaller (1.1 x 10(3) M(-1) s(-1)) than the value obtained at pH* 7.6 (3.7 x 10(3) M(-1) s(-1)). The effect on the self-exchange reactivity is mainly due to an alteration in the reorganization energy of the copper site brought about by the structural change resulting from His6 protonation.
Sato
Katsuko
K
Department of Chemistry, University of Newcastle upon Tyne, Newcastle upon Tyne, NE1 7RU, UK.
Dennison
Christopher
C
D000421:Alcaligenes / Q000737:chemistry*; D001400:Azurin / Q000031:analogs & derivatives* / Q000737:chemistry*; D001665:Binding Sites; D003300:Copper / Q000737:chemistry; D004579:Electron Transport; D006639:Histidine / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D008024:Ligands; D009682:Magnetic Resonance Spectroscopy; D010084:Oxidation-Reduction; D011522:Protons; D013053:Spectrophotometry
D008024:Ligands; D011522:Protons; C047163:pseudoazurin; D001400:Azurin; D006639:Histidine; D003300:Copper