title: Ligand binding and protein dynamics in cupredoxins.
abstract: Type 1 copper sites bind nitric oxide (NO) in a photolabile complex. We have studied the NO binding properties of the type 1 copper sites in two cupredoxins, azurin and halocyanin, by measuring the temperature dependence of the ligand binding equilibria and the kinetics of the association reaction after photodissociation over a wide range of temperature (80-280 K) and time (10(-6)-10(2) s). In both proteins, we find nonexponential kinetics below 200 K that do not depend on the NO concentration. Consequently, this process is interpreted as geminate recombination. In azurin, the rebinding can be modeled with the Arrhenius law using a single pre-exponential factor of 10(8.3) s-1 and a Gaussian distribution of enthalpy barriers centered at 22 kJ/mol with a width [full width at half-maximum (FWHM)] of 11 kJ/mol. In halocyanin, a more complex behavior is observed. About 97% of the rebinding population can also be characterized by a Gaussian distribution of enthalpy barriers at 12 kJ/mol with a width of 6.0 kJ/mol (FWHM). The pre-exponential of this population is 1.6 x 10(12) s-1 at 100 K. After the majority population has rebound, a power-law phase that can be modeled with a gamma-distribution of enthalpy barriers is observed. Between 120 and 180 K, an additional feature that can be interpreted as a relaxation of the barrier distribution toward higher barriers shows up in the kinetics. Above 200 K, a slower, exponential rebinding appears in both cupredoxins. Since the kinetics depend on the NO concentration, this process is identified as bimolecular rebinding.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Department of Physics, University of Illinois at Urbana-Champaign 61801-3080, USA.
authors.forename: D
authors.initials: D
authors.lastname: Ehrenstein
authors.forename: M
authors.initials: M
authors.lastname: Filiaci
authors.forename: B
authors.initials: B
authors.lastname: Scharf
authors.forename: M
authors.initials: M
authors.lastname: Engelhard
authors.forename: P J
authors.initials: PJ
authors.lastname: Steinbach
authors.forename: G U
authors.initials: GU
authors.lastname: Nienhaus
mesh_terms: D001400:Azurin / Q000031:analogs & derivatives* / Q000378:metabolism* / Q000528:radiation effects; D001425:Bacterial Outer Membrane Proteins / Q000378:metabolism* / Q000528:radiation effects; D001665:Binding Sites; D003300:Copper; D007700:Kinetics; D008024:Ligands; D008027:Light; D008667:Metalloproteins / Q000378:metabolism* / Q000528:radiation effects; D008956:Models, Chemical; D008968:Molecular Conformation; D009569:Nitric Oxide / Q000378:metabolism*; D013053:Spectrophotometry; D013696:Temperature; D013816:Thermodynamics
chemical_list: D001425:Bacterial Outer Membrane Proteins; D008024:Ligands; D008667:Metalloproteins; C050107:cupredoxin; C084860:halocyanin; D001400:Azurin; D009569:Nitric Oxide; D003300:Copper