Complexes of transition metals and amino acid residues play important roles in many biological processes, such as electron transfer, redox and catalysis.^[1] In these processes, the enzyme active sites are highly specific and often involve histidine (His) residues interacting with divalent transition metal cations, i.e., Mn²⁺, Fe²⁺, Co²⁺, Ni²⁺, Cu²⁺, and Zn²⁺.^[2] Furthermore, these metals are the essential trace elements for living bodies. The deficiencies of them will cause diseases and a supplementation is needed. Typically, the deficiency of Cu will cause the Menkes disease, a rare genetic disorder. Subcutaneous administration of the Cu²⁺/His complex is the only promising way to treat this disease.^[3–6] To study the function of Cu²⁺/His complex in Cu transport, it has already been characterized by many physical techniques, such as infrared, Raman, extended x-ray absorption fine structure (EXAFS) since it was discovered in human blood in 1966.^[7,8]

The molecule formula of His is C₆H₉N₃O₂, including one imidazole ring. There are several potential coordination sites for metal ions to coordinate the His molecule, e.g. the N atom from amino (N_am), the O atom from carboxyl (O_carboxyl) and the two N atoms from imidazole (they are both named N_im, the one near C_β is N^π, the other one is N^τ). So the structure of Cu²⁺/His complex is complicated. The results obtained by other work suggest that Cu²⁺/His complex keeps an octahedral configuration with O/N atoms from water molecules and His coordinated with Cu ion.^[8] The details regarding the three-dimensional (3D) local structure of Cu²⁺/His in solutions with different pH values was not yet investigated.

X-ray absorption fine structure (XAFS) is a powerful technique to detect the local structure information about metal ions at high spatial resolution, which has been successfully used in lots of research fields.^[9–15] It is composed of two parts, i.e., EXAFS and x-ray absorption near-edge structure (XANES). XANES is more sensitive to the local structure configuration and coordination atoms around metal atoms than EXAFS. In order to make a further study, the structures of Cu²⁺/His complexes at three typical pH values (2.5, 6.5, and 8.5) are studied by both qualitative and quantitative XANES analysis. In the quantitative analysis, MINUIT XANes (MXAN) that employs the full multiple scattering (MS) approach to a description of the XANES region was used.^[16–19] MXAN has been proved to be powerful in a number of interesting situations.^[20–24] It can be employed to reveal more detailed high resolution 3D structures of the studied systems with a lower command for S/N than EXAFS.

Solutions of Cu²⁺/His with a molar ratio of 1:2 at pH values of 2.5, 6.5, 8.5 were prepared by dissolving 0.01-Mol L-histidine and 0.005-Mol CuCl₂·2H₂O into the de-ionized water with a volume of 40 ml. Then NaOH or HCl solution was added to adjust the pH value.

XAFS measurements were performed at the XAFS station of 1W1B beamline of the Beijing Synchrotron Radiation Facility (BSRF). The typical energy of the storage ring was 2.5 GeV with a current decreasing from 250 mA to 160 mA during experiment runs. An Si (111) double crystal monochromator was used to minimize the high harmonics content through the detuning of the two crystals by 20%. The absolute energy position was calibrated using a Cu metal foil. The XAFS spectra of Cu K-edge of Cu²⁺/His at different pH values were recorded in the fluorescence mode. For the experiments, the solution sample was kept in a sample cell of polyethylene. An ion chamber which 50% argon and 50% nitrogen flowed into was used to monitor the incident beam intensity (I₀) while a Lytle detector which pure Ar flowed into was used to record the fluorescence signal (I_f). Data of all the experimental XAFS spectra were analyzed by using WinXAS3.1^[25] and the MXAN package.^[16–19] The initial models of Cu²⁺/His complexes at different pH values for XANES analysis were established according to the results suggested by other works.^[8]

In this work, the local structural configurations of Cu²⁺/His complexes at different pH values are performed by using the XANES spectroscopy. The 3D stereo structural information is extracted by fitting the near-edge spectra. The geometric structure becomes disordered in basic solution, resulting in the variations of coordinates and bond distances. This work is not only complementary to a general understanding of the structure in Cu²⁺/His complex, but also facilitates our understanding of the physiological mechanisms of Cu²⁺/His in biological systems.