Magnetic susceptibility studies of laccase and oxyhemocyanin.

Abstract

(1,2), and the oxidases laccase, ascorbate oxidase, and ceruloplasmin (3). Additionally cytochrome c oxidase has been proposed to contain a coupled heme ironcopper pair as the site of dioxygen binding and reduction (4). In no case has any electron paramagnetic resonance (EPR) signal attributable to a binuclear site been observed in the native protein (5). Although other structural possibilities are consistent with this observation, it is generally thought that the metal ions involved are strongly antiferromagnetically coupled, resulting in a diamagnetic or even spin ground state (5, 6). The temperature dependence of the magnetic susceptibility of such systems is distinctive and may be used to distinguish antiferromagnetic coupling from a truly diamagnetic structure-e.g., a Cu(I) dimer. For two antiferromagnetically coupled S = 1/2 Cu(II) ions, the susceptibility will be a maximum at a temperature that is simply related to the energy difference, J, between the diamagnetic ground state and the paramagnetic excited state (7). Antiferromagnetic coupling between two copper ions has been observed in a wide variety of dimeric Cu(II) complexes (8) and in the four-copper derivative of bovine superoxide dismutase (9). We have focused our efforts on oxyhemocyanin and laccase. It is generally accepted that the dioxygen binding site in oxyhemocyanin is a binuclear copper(II) peroxo complex, the evidence coming mainly from resonance Raman (10) and other spectroscopic (11,12) experiments. The four copper atoms in laccase are distributed in types 1 (paramagnetic), 2 (paramagnetic), and 3 (binuclear; EPR nondetectable) sites. The type 3 coppers apparently function as a two-electron acceptor and are thought to be the site of interaction with dioxygen (3).Previous magnetic susceptibility measurements showed oxyhemocyanin to be diamagnetic over the temperature range 35-250 K (13,14). Over this same range, the susceptibility of Rhus laccase showed, in addition to the Curie law paramagnetism of copper types 1 and 2, non-Curie paramagnetism at temperatures above 80 K that was consistent with an antiferromagnetically coupled Cu(II) pair with J = 170 ± 30 cm1 (13).Owing to the fact that the fit of theory to the observed deviation from Curie law in laccase was not entirely satisfactory (due in part to the low precision of the measurements) and realizing the importance of these observations to understanding the copper-site electronic structure of hemocyanin and laccase, we have remeasured the susceptibilities over a wider temperature range by using susceptometers recently developed at SHE Corporation, San Diego, CA. Although oxyhemocyanin was again observed to be essentially diamagnetic over the entire temperature range, laccase, in contrast to our previous results, displayed only the Curie law behavior of copper types 1 and 2 over the temperature range examined. MATERIALS AND METHODSRhus vernidfera laccase was purified by the method of Reinhammar (15) from the acetone powder (Saito & Co., Ltd., Japan) to a A2o/A614 value <15. A sample w...

Copper complexes of polyfunctional phthalazines. Mononuclear and polynuclear derivatives

“…tyrosinase, laccase. Magnetic measurements on protein extracts indicate strong antiferromagnetic exchange between the copper-(11) centres in Rhus vernicifera laccase (-25 2 1000 cm-I) (36) and in oxyhemocyanin (Megathura crenulata; -2J 2 1250 cm-I) (37). Strong antiferromagnetic exchange is observed in binuclear copper systems involving single hydroxy bridges (29,35,38) and exchange increases with bridge angle (29,34).…”

Crystal structure of deoxygenated limulus polyphemus subunit II hemocyanin at 2.18 Å resolution: Clues for a mechanism for allosteric regulation

“…In addition, the cupric ions must be strongly antiferromagnetically coupled by the bound peroxide, since oxygenated hemocyanins are EPR silent (Dooley et al, 1978). Based on these data several models for the oxygenated dinuclear copper site in hemocyanin have been proposed (Eickman et al, 1979;Maddaluno & Giessner-Prettre, 1991;Ross & Solomon, 1991).…”

Structure and magnetism in a hydroxy bridged binuclear copper(II) system with a "tunable" binuclear centre

“…Magnetic measurements on protein extracts indicate strong antiferromagnetic exchange between the copper(I1) centres in Rhus vernicifera laccase (5) (-2J > 1000 cm-') and in oxyhemocyanin (Megathura crenulata; -2 J > 1250 cm-') (5,6). Strong antiferromagnetic exchange is observed in binuclear copper(I1) systems involving single hydroxy bridges (7)(8)(9) and exchange increases with increasing bridge angle (7).…”