Reactivity, Electrochemical, and Spectroscopic Studies of Type 2 Copper-Depleted Rhus vernicifera Lacease

Spectroelectrochemical, circular dichroism (CD), and kinetic measurements have been performed on native and type 2 copper-depleted (T2D) tree laceases to determine the effect of type 2 copper removal on the tertiary structure, blue copper reduction potential, and electron-transfer reactivity of the metalloenzyme. The midpoint potentials (E°) of type 1 Cu in T2Dr (reduced type 3 center) and native laceases, determined with (hydroxyethyl)ferrocene (HEF) as mediator, are essentially identical (430 and 429 mV, respectively; 25 °C, pH 7.0, I = 0.5 M). Comparisons of the 300-700-nm CD spectra of native, T2Dr, and T2D0X (oxidized type 3 center) laceases reveal no detectable structural change at the type 1 copper center associated with removal of the type 2 Cu atom and only small effects of the oxidation state of the type 3 center on the environment of the type 1 site. Stopped-flow kinetic studies of electron transfer to T2Dr-type 1 Cu(II) from HEF and seven hydroquinones were performed to verify the importance of type 2 copper in the binding of polyphenolic substrates and to determine the effect of type 2 copper removal on the intrinsic electron-transfer reactivity of the blue copper atom. Removal of type 2 copper has remarkably little effect on the overall electron-transfer reactivity of substituted hydroquinones with lacease type 1 Cu(II), as measured by a second-order rate constant (k). The reduction rate of type 1 copper is slightly sensitive to the oxidation state of the type 3 center, as k values for electron transfer to T2Dr and T2D0X from hydroquinone are 5.2 X 102 and 3.2 X 102 M^-1 s^-1, respectively, at 25 °C, pH 7.0, and / = 0.5 M. Detailed comparisons of rate-substrate concentration profiles show that ES complex formation constants are much smaller in T2Dr than in native lacease. The role of type 2 copper as a substrate binding site in the blue copper reduction pathway therefore is confirmed. The intrinsic electron-transfer reactivity of lacease type 1 copper increases substantially upon removal of the type 2 copper atom, as shown by rate constants for the HEF reduction of T2Dr-type 1 Cu(II) (1.9 X 104 M^-1 s^-1) and native type 1 Cu(II) (4.8 X 103 M^-1 s^-1), at 25 °C, pH 7.0, and I = 0.5 M. Apparent blue copper self-exchange electron-transfer rate constants estimated from the application of relative Marcus theory to these HEF rate data are 1.6 and 2.5 X 101 M^-1 s^-1 for the native and T2D proteins, respectively.