Kinetic, spectroscopic, and structural (extended X-ray absorption fine structure) studies on the type 1 blue copper protein umecyanin

Abstract

The effects of pH variations on the visible spectrum (pK_a 9.46 ± 0.04), e.s.r. spectrum, extended X-ray absorption fine structure (EXAFS), and on the reactivity of the blue copper protein umecyanin have been investigated. The electron-transfer reactions of umecyanin have been studied with the complexes [Co(dipic)₂]_–(dipic = dipicolinate) and [Co(C₂O₄)₃]^3– as oxidants for UCu(I), and [Ru(NH₃)₅(py)]²⁺(py = pyridine) as a reductant for UCu(II), at 25 °C, I= 0.10 M (NaCl). The oxidation of umecyanin with [Fe(CN)₆]^3– is extremely rapid, consequently a limited study was carried out at a lower temperature. The oxidation of the protein by [Co(phen)₃]³⁺(phen = 1,10-phenanthroline) was also studied under these conditions for comparison. Of interest are the pK_a values observed for the oxidation of umecyanin by [Co(C₂O₄)₃]^3–(pK_a 9.68 ± 0.08) and the reduction of umecyanin by [Ru(NH₃)₅(py)]²⁺(pK_a 9.50 ± 0.07). The low temperature (20 K)X-band e.s.r. spectrum of oxidised umecyanin (g_‖= 2.32, g_⊥= 2.06) shows little variation over the pH range 7.5–10.5. The Cu K-edge EXAFS of umecyanin in both oxidation states and at pH 7.5 and 10.5 were obtained. Analysis of data from the oxidised protein shows the Cu atom has two nitrogen ligands at 1.99 ± 0.03 Å and one sulphur at 2.13 ± 0.02 Å. In the reduced protein the bond lengths increase to 2.03 ± 0.03 Å and 2.21 ± 0.02 Å respectively. There was no detectable difference in co-ordination number or bond lengths at the different pH values, and no fourth ligand could be detected by the EXAFS technique. The results from these various studies are discussed in terms of changes at or near the active site of umecyanin.