Analysis of the redox reaction of an archaebacterial copper protein, halocyanin, by electrochemistry and FTIR difference spectroscopy.
Halocyanin is a recently discovered archaebacterial copper protein classified as "type I" small blue copper protein (Scharf, B., Ph.D. Thesis, University of Bochum, Germany). Its redox properties were investigated by a combination of protein electrochemical and spectroscopic techniques. Using electrochemical reactions in an ultrathin-layer electrochemical cell developed for UV/vis and IR spectroscopy, halocyanin could be quantitatively and reversibly oxidized and reduced. The titration of the absorption band at 600 nm can be perfectly described by a Nernst curve with n = 1 electron transferred; a quantitative fit yields a midpoint potential, Em, of 183 mV (vs SHE) at a pH of 7.3. The midpoint potential falls constantly from +333 mV at pH 4 to +119 mV at pH 10, with three regions around pH 4.5, 6.5, and 8.5 where the pH dependence is ca. -60 mV/pH unit, indicating the uptake of a proton with the reduction. By analogy with other small type I copper proteins, the three pK values suggested by the pH dependency of Em might be associated with three histidines which interact with the redox site. Electrochemically induced reduced-minus-oxidized Fourier transform infrared difference spectra in the 1800-1000 cm-1 range at neutral pH show a number of strong difference bands between ca. 1700 and 1600 cm-1 as well as smaller difference structures between 1600 and 1200 cm-1. The maximum amplitude of the difference bands--only ca. 1% of the amide-I absorption at ca. 1639 cm-1--indicates that only small protein rearrangements occur upon the redox transition.(ABSTRACT TRUNCATED AT 250 WORDS)
Brischwein
M
M
Institut für Biophysik und Strahlenbiologie, Universität Freiburg, Germany.
Scharf
B
B
Engelhard
M
M
Mäntele
W
W
D001105:Archaea / Q000737:chemistry*; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry*; D001665:Binding Sites; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D003300:Copper / Q000378:metabolism; D004563:Electrochemistry; D006863:Hydrogen-Ion Concentration; D008667:Metalloproteins / Q000737:chemistry*; D010084:Oxidation-Reduction; D011487:Protein Conformation; D017550:Spectroscopy, Fourier Transform Infrared
D001425:Bacterial Outer Membrane Proteins; D008667:Metalloproteins; C084860:halocyanin; D003300:Copper