Spectroscopic and Magnetic Studies of the Coordination Environment and Electronic Structure of Copper Sites in Blue Copper Oxidases

Low temperature absorption, circular dichroism, and magnetic circular dichroism measurements have been made on the multicopper oxidases Rhus vernicifera laccase, Polyporus versicolor laccase, and human ceruloplasmin. Near infrared bands have been observed that are assigned to ligand field transitions of the type 1 (blue) Cu(II). The low energies of these bands, when considered together with the EPR g values, are consistent only with a tetragonally (D2d) distorted tetrahedral geometry for type 1 Cu(II). Analysis and comparison of data obtained on the blue oxidases and single blue copper proteins indicate that three kinds of blue copper may be distinguished spectroscopically and electrochemically. No evidence of inequivalent type 1 sites was found in native or azide inhibited ceruloplasmin. Modified forms of the laccases and ceruloplasmin, in which the type 1 copper is specifically reduced or removed, were also investigated. Absorption and circular dichroism bands were observed that are attributed to the types 2 and 3 copper ions. Distorted tetrahedral geometry may be ruled out for these sites as no near infrared bands were present. Observed transitions are consistent with tetragonal six, five or square planar four coordination. Superhyperfine in the EPR spectra of modified ceruloplasmins established three or four nitrogen atoms as ligands of t he type 2 Cu(II). Binding of up to two fluoride ions by ceruloplasmin type 2 copper was also demonstrated . Magnetic susceptibility measurements on Rhus vernicifera laccase showed the type 3 coppers to be diamagnetic from 5-260 K. If the type 3 site is formulated as an antiferromagnetically coupled Cu(II) pair then J ≥ 500 cm-1. Data were also obtained on Limulus polyphemus oxyhemocyanin which require a lower limit on J of 550 cm-1. Curie law behavior of the susceptibility of Rhus laccase at low temperatures indicates that types 1 and 2 copper must be separated by several angstroms and are not bridged by a common ligand(s) in the native protein. However, binding of fluoride at the type 2 site in both Rhus and Polyporus laccase perturbs the type 1 circular dichroism spectrum. This may reflect catalytically significant conformational changes designed to facilitate intramolecular electron transfer.