Structural changes caused by radiation-induced reduction and radiolysis: the effect of X-ray absorbed dose in a fungal multicopper oxidase

Abstract

X-ray radiation induces two main effects at metal centres contained in protein crystals: radiation-induced reduction and radiolysis and a resulting decrease in metal occupancy. In blue multicopper oxidases (BMCOs), the geometry of the active centres and the metal-to-ligand distances change depending on the oxidation states of the Cu atoms, suggesting that these alterations are catalytically relevant to the binding, activation and reduction of O(2). In this work, the X-ray-determined three-dimensional structure of laccase from the basidiomycete Coriolopsis gallica (Cg L), a high catalytic potential BMCO, is described. By combining spectroscopic techniques (UV-Vis, EPR and XAS) and X-ray crystallography, structural changes at and around the active copper centres were related to pH and absorbed X-ray dose (energy deposited per unit mass). Depletion of two of the four active Cu atoms as well as low occupancies of the remaining Cu atoms, together with different conformations of the metal centres, were observed at both acidic pH and high absorbed dose, correlating with more reduced states of the active coppers. These observations provide additional evidence to support the role of flexibility of copper sites during O(2) reduction. This study supports previous observations indicating that interpretations regarding redox state and metal coordination need to take radiation effects explicitly into account.

Figure 1

The Cg L fold, showing the copper-centre distribution for the three cupredoxin domains (domain 1, grey; domain 2, green; domain 3, purple) and the dioxygen-entrance (red) and water-exit (blue) channels.

Figure 2

(a) CW EPR spectra collected from Cg L solutions at different pH values (red, green and blue correspond to pH 4.0, 5.5 and 7.0, respectively, while 1a and 1b refer to experimental data and simulation, respectively). (b) Magnification of the T2 Cu region showing the pH-induced changes caused at this metal centre.

Figure 3

Active crystals of Cg L grown in 5% PEG 1000, 5% PEG 8000, and 100 mM MES pH 4.0: (a) 2 min after soaking in 2 mM DMP, (b) 30 min after soaking, (c) 60 min after soaking.

Figure 4

Alternative conformation of the T2 Cu-coordinating His418 fitted into positive density in the F _o − F _c map (green). 2F _o − F _c = 1.5σ (blue) and F _o − F _c = 3.5σ (green) electron densities are displayed and correspond to the crystal at pH 5.5.

Figure 5

Structural differences observed in Cg L crystals irradiated at different pH values. Differences at T1 Cu are shown in (a) and (b), and differences observed at the TNC (T2 Cu/T3 Cu) are displayed in (c) and (d). (a, c) pH 4.0, (b, d) pH 5.5.

Figure 6

Anomalous map showing asymmetry in the electron density at T3 Cu_A and T3 Cu_B. The map (magenta) is contoured at 0.7 e Å⁻³. Data were obtained at NSLS at an incident wavelength of 1.3778 Å.

Figure 7

X-ray absorption spectra showing the reduction of Cu^II to Cu^I upon X-ray irradiation. The oxidized spectrum (solid line) corresponds to Cg L crystals before X-ray data collection, while the reduced spectrum (dotted line) shows that reduction occurs during collection of a complete data set.

Figure 8

The pH dependence of the catalytic potential (E _cat) and its relation to the activity of Cg L is shown.