Kinetics and mechanism of the acid transition of the active site in plastocyanin.
Exchange on the microsecond time scale between the protonated and deprotonated forms of His92 in the copper site of reduced plastocyanin from the cyanobacteria Anabaena variabilis was monitored using 15N NMR relaxation measurements. On the basis of the dependence of the kinetics on pH and phosphate buffer concentration, we propose a two-step model for the protonation of the copper site in agreement with previous crystallographic studies. It is shown that the proton transfer is the rate-limiting step in the reaction at low buffer concentrations, whereas at high buffer concentrations, another step becomes rate-limiting. We suggest that the latter step is a concerted dissociation of His92 from the Cu(I) ion and a 180 degrees rotation of the imidazole ring, which precede the protonation. The first-order rate constant for the dissociation of His92 from the Cu(I) ion is estimated to be 2.4 x 10(4) s(-1). Also, a cooperative effect of the protonation of the remote His61 on the protonation of His92 and the redox properties of the protein was investigated by substituting His61 with asparagine. The mutation causes a modest change in both the pKa value of His92 and the redox potential of the protein.
Hass
Mathias A S
MA
Department of Chemistry, University of Copenhagen, Universitetsparken 5, DK-2100 Copenhagen Ø, Denmark.
Christensen
Hans E M
HE
Zhang
Jingdong
J
Led
Jens J
JJ
D017033:Anabaena / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D001665:Binding Sites; D003300:Copper / Q000737:chemistry / Q000378:metabolism; D006639:Histidine / Q000737:chemistry / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D019906:Nuclear Magnetic Resonance, Biomolecular; D010970:Plastocyanin / Q000737:chemistry* / Q000378:metabolism; D011522:Protons
D001426:Bacterial Proteins; D011522:Protons; D006639:Histidine; D003300:Copper; D010970:Plastocyanin