Structures of Cu(II)-(histidine) 2 in solution

Abstract

We have examined the structure of Cu(II)-(histidine) 2 in solution using optical, electron paramagnetic resonance (EPR) and electron spin-echo (ESE) spectroscopies. Histidine is a potential tridentate ligand with three groups capable of binding Cu(II): 1) carboxyl oxygen 2) imidazole nitrogen and 3) amino nitrogen. Histidine is involved in the coordination of metal ions in a number of copper proteins, including superoxide dismutase, ceruloplasmin, ascorbate oxidase, galactose oxidase, etc. In addition, histidine has been implicated in the in vivo transport of copper between albumin and cells. Previously the structure of Cu(II)-(histidine) 2 was determined using proton NMR and optical spectroscopies, and X-ray crystallography. Two structures were suggested. In the first, the Cu(II) is coordinated by two imidazole and by two amino nitrogen atoms. In the second structure the Cu(II) is ligated to a single imidazole nitrogen, and an amino nitrogen from one of the histidines and an amino nitrogen and a carboxyl oxygen from the second histidine. Computer aided analysis of the optical pH titration of Cu(II)-(histidine) 2, at 25 °C, shows two transitions, with pK's of 3.6 and 5.5. Both titrations have n= 1. An EPR pH titration at 77 K, shows 2 transitions with pK's of 2.8 and 4.4 and n values of 2 and 1, respectively. The lower pK represents the complexation of Cu(II)-aquo (g ∥ = 2.417, A ∥ = 14.1 mK) by histidine to form an intermediate complex (g ∥ = 2.306, A ∥ = 18.6 mK). The second pK represents the conversion of this intermediate to the form of the complex present at the physiological pH (g∥ = 2.242, A∥ = 18.8 mK). The changes in g∥ suggest the binding of an additional equatorial nitrogen when the intermediate is converted to the neutral pH form. In a separate study, we used ESE spectroscopy to determine the number of imidazole nitrogen atoms equatorially coordinated Cu(II) in the bis histidine complex. The observed periodicities in the ESE decay envelope are due to interactions of the unpaired electron with the remote, protonated 14N of bound imidazole. Directly coordinated 14N is not observed in these experiments. Fourier transformation of the modulation pattern for the complex prepared at pH 7.6 shows frequencies at 0.7, 1.5, and 4.0 MHz, which are characteristic of Cu(II) coordinated by imidazole. The depth of the modulation pattern has been shown to be a product function of the number of interacting nuclei and their distance. Thus if two imidazoles are coordinated to Cu(II), the depth of modulation is the square of that seen for a single coordinated imidazole provided that the CuN bond lengths are the same. Using Cu(II)diethylenetriamineimidazole, and Cu(II)oxalate(imidazole) 2 as a single, and double imidazole models, we can quantitate the number of imidazoles bound to Cu(II) in Cu(II)-(histidine) 2. The depth of modulation for Cu(II)(histidine) 2 is comparable to that observed for Cu(II)diethylenetriamineimidazole. Therefore, at physiological pH, a single imidazole is equatorially coordinated to the metal ion. At pH 3.4, where the predominant species are Cu(II)-aquo and the low pH intermediate, the modulation pattern characteristic of coordinated imidazole is still observed. Thus imidazole remains bound to Cu(II) here as well.