Towards accurate structural characterization of metal centres in protein crystals: the structures of Ni and Cu T₆ bovine insulin derivatives

The level of structural detail around the metal sites in Ni²⁺ and Cu²⁺ T₆ insulin derivatives was significantly improved by using a combination of single-crystal X-ray crystallography and X-ray absorption spectroscopy. Photoreduction and subsequent radiation damage of the Cu²⁺ sites in Cu insulin was followed by XANES spectroscopy. Using synchrotron radiation (SR), the crystal structures of T₆ bovine insulin complexed with Ni²⁺ and Cu²⁺ were solved to 1.50 and 1.45 Å resolution, respectively. The level of detail around the metal centres in these structures was highly limited, and the coordination of water in Cu site II of the copper insulin derivative was deteriorated as a consequence of radiation damage. To provide more detail, X-ray absorption spectroscopy (XAS) was used to improve the information level about metal coordination in each derivative. The nickel derivative contains hexacoordinated Ni²⁺ with trigonal symmetry, whereas the copper derivative contains tetragonally distorted hexacoordinated Cu²⁺ as a result of the Jahn–Teller effect, with a significantly longer coordination distance for one of the three water molecules in the coordination sphere. That the copper centre is of type II was further confirmed by electron paramagnetic resonance (EPR). The coordination distances were refined from EXAFS with standard deviations within 0.01 Å. The insulin derivative containing Cu²⁺ is sensitive towards photoreduction when exposed to SR. During the reduction of Cu²⁺ to Cu⁺, the coordination geometry of copper changes towards lower coordination numbers. Primary damage, i.e. photoreduction, was followed directly by XANES as a function of radiation dose, while secondary damage in the form of structural changes around the Cu atoms after exposure to different radiation doses was studied by crystallography using a laboratory diffractometer. Protection against photoreduction and subsequent radiation damage was carried out by solid embedment of Cu insulin in a saccharose matrix. At 100 K the photoreduction was suppressed by ∼15%, and it was suppressed by a further ∼30% on cooling the samples to 20 K