Proton and Oxygen-17 Magnetic Resonance Relaxation in Rhus Laccase Solutions: Proton Exchange with Type 2 Copper(II) Ligands

Longitudinal (T₁ ^-1) and transverse (T₂ ^-1) nuclear magnetic resonance (NMR) relaxation rates of ¹H and ¹⁷O of water molecules were measured in solutions of oxidized Rhus vernicifera laccase in order to investigate the structural and dynamic aspects of water coordination to its type 2 Cu(II) site. The magnitude and negative temperature dependence of the paramagnetically induced proton T₁ ^-1 and T₂ ^-1 showed that the proton fast-exchange mechanism is operative. The field dependence of T_{1, pmg} ^-1 gave an apparent correlation time of 2.7 ns for the 9-60-MHz range. T_{1, pmg} ^-1 is pH dependent, giving two dissociation steps with pK = 6.2 and 8.6 which are attributed to the hydrolysis of a H₂O molecule and a protein hydroxyl group coordinated to the type 2 Cu(II), respectively. Relaxation rates of ¹⁷O were not paramagnetically enhanced. Neither T₁ ^-1 nor T₂^-1 values of both ¹H and ¹⁷O were significantly affected by the binding of fluoride or azide ions to type 2 Cu(II). Our data lead to the following interpretation: The water oxygen equatorially bound to type 2 copper does not exchange on the NMR time scale. The paramagnetic relaxation via the fast exchange mechanism originates from proton transfer between the Cu(II)-bound water molecule or hydroxyl group and the buffered solution, acid-base catalyzed by a protein residue. The relative inertness of the water oxygen is consistent with the structural concept of a cavity which accommodates the laccase type 2 (and type 3) copper sites, the active centers involved in O₂ reduction, and is connected to the bulk solvent via an orifice which in the native oxidized enzyme is only penetrable by protons. A tentative model of the type 2 Cu coordination structure is presented.