No abstract

Near-UV absorption studies have been carried out for various metal-replaced derivatives of spinach plastocyanin. Above 250 nm plastocyanin derivatives containing Zn(II), Cd(II), or Cu(II), as well as apoplastocyanin, exhibit very similar absorptions due to phenylalanine and tyrosine residues. In contrast, forms containing Hg(II), Cu(I), or Ag(I) show enhanced absorbance in the UV region. In difference spectra with apoplastocyanin as a reference, the Hg(II) derivative exhibits a maximum around 247 nm (Ae -9800 ± 100 M'1 cm'1) and a shoulder at 280 nm (Ae = 2100 M"1 cm'1) while the Cu(I) analogue has a strong absorbance at 275 nm (Ae ^3320 ± 20 M"1 cm"1) and a weaker shoulder at 310 nm. In Ag(I) plastocyanin a band is apparent at 240 nm (Ae 5100 ± 100 M'1 cm'1). The metal dependence of the absorption spectrum suggests that the metal centers are directly involved in the transitions, and literature data support this view. For the Hg(II) derivative the bands can be assigned as S(Cys) -» Hg(II) charge-transfer absorptions analogous to transitions found in mercury thioneins. For the Cu(I) and Ag(I) derivatives the bands are plausibly assigned to metal-centered or charge-transfer transitions involving the radial extension of electron density from the zid shell. A similar assignment is advanced for bands, previously assigned as ligand-to-metal charge-transfer transitions, in the spectra of copper thioneins. These results show that UV spectra can sometimes be useful in characterizing protein derivatives containing metal ions that, on the basis of visible spectra, have been regarded as spectroscopically "silent".

A simple colorimetric test for the Cu(I) content in blue copper proteins is described. The procedure is based on the formation of a complex between Cu(I) and 2,2'-biquinoline in an acetic acid medium. Analyses of spinach plastocyanin, Pseudomonas aeruginosa azurin and Rhus vernicifera stellacyanin show that the cysteine residue in the type 1 site does not induce Cu(II) reduction under our conditions. There is evidence in laccase samples for the presence of an endogenous reductant that can reduce 0.14 +/- 0.04 mol of Cu(II)/mol of protein; however, the addition of EDTA eliminates the interference. The analysis shows that 25 +/- 2% of the type 3 copper ions are in the reduced form in the resting enzyme and that 80 +/- 15% of the type 3 copper ions are reduced in preparations of type-2-depleted laccase. There is growing interest in the development of chemically modified forms of laccase, and our method should be very useful for establishing the valence state of the metal centres in the various derivatives.

No abstract

No abstract