Optical spectroscopic investigation of the alkaline transition in umecyanin from horseradish root.
Umecyanin (UMC) from horseradish root belongs to the stellacyanin subclass of the phytocyanins, a family of plant cupredoxins. The protein possesses the typical type-1 His(2)Cys equatorial ligand set at its mononuclear copper site but has an axial Gln ligand in place of the usual weakly coordinated Met of the plantacyanins, uclacyanins, and most other cupredoxins. UMC exhibits, like other phytocyanins, altered visible, EPR, and paramagnetic (1)H NMR spectra at elevated pH values and also a modified reduction potential. This alkaline transition occurs with a pK(a) of approximately 10 [Dennison, C., Lawler, A. T. (2001) Biochemistry 40, 3158-3166]. In this study, we investigate the alkaline transition by complementary optical spectroscopic techniques. The contemporary use of absorption, fluorescence, dynamic light scattering, and resonance Raman spectroscopy allows us to demonstrate that the alkaline transition induces a reorganization of the protein and that the overall size of UMC increases, but protein aggregation does not occur. The transition does not have a dramatic influence on the active-site environment of UMC, but there are subtle alterations in the Cu site geometry. Direct evidence for the strengthening of a Cu-N(His) bond is presented, which is in agreement with the hypothesis that the deprotonation of the N(epsilon2)H moiety of one of the His ligands is the cause of the alkaline transition. A weakening of the Cu-S(Cys) bond is also observed which, along with a weakened axial interaction, must be due to the enhanced Cu-N(His) interaction.
Delfino
Ines
I
Biophysics and Nanoscience Centre, INFM-CNISM, Dipartimento Scienze Ambientali, Università della Tuscia, Largo dell'Università, I-01100 Viterbo, Italy.
Sato
Katsuko
K
Harrison
Mark D
MD
Andolfi
Laura
L
Bizzarri
Anna Rita
AR
Dennison
Christopher
C
Cannistraro
Salvatore
S
D031215:Armoracia / Q000737:chemistry*; D003300:Copper / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D008667:Metalloproteins / Q000737:chemistry*; D008958:Models, Molecular; D009584:Nitrogen / Q000737:chemistry; D018517:Plant Roots / Q000737:chemistry*; D017434:Protein Structure, Tertiary; D013455:Sulfur / Q000737:chemistry
D008667:Metalloproteins; C002054:umecyanin; D013455:Sulfur; D003300:Copper; D009584:Nitrogen