81 zyxwvuts Inorganica Chimica Acta, 91 (1984) 81- 88 Peroxide and.Redox Titrations of Type 2 Copper Depleted Lactase PATRICK FRANK*, OLE FARVER and ISRAEL PECHT Department of Chemical Immunology, The W eizmann Institute of Science, Rehovot 76100, Israel The chemistry of Type 2 copper depleted T2D Rhus lactase has been investigated with regard to the binding of peroxide, and the ability of the enzyme to undergo reduction and reoxidation. Although the peroxide affinity is diminished in the T2D enzyme (1 O4 M- ‘} relative to the holo-enzyme (> 10’ M- ‘) the actual mode of binding as a Type 3 u-peroxo complex remains, as indicated by absorption and CD spectral measurements. Anaerobic reductive and reoxidative titrations with hydroquinone and hydro- gen peroxide respectively revealed that the Type 3 copper pairwise interaction is disrupted during reduc- tion but can be restored on reoxidation. The concept of separate Type 2 and Type 3 copper redox centers is suggested to be inadequate in view of the loss of functional integrity by the Type 3 site on removal of Type 2 copper. (Abbreviations used in the Text: U. K: ultraviolet, CD: circular dichroism, T2D lactase: Rhus lactase which has been depleted of Type 2 copper, H2Q: hy droquinone, EPR: electron paramagnetic reso- nance. ). The controlled catalytic utilization of ambient dioxygen in cool, dilute, neutral aqueous solu- tions ranks, along with nitrogen fixation, hydro- genation and photosynthesis, as one of the spectac- ular achievements of evolutionary invention. Among the biochemical constructsenzymes, which manage and direct the powerful reactivity of dioxygen, considerable attention has been focused on the oxidases. In particular, cytochrome oxidase, a mam- malian 2iron/2copper enzyme, and the ‘blue’ copper oxidases derived from both plant and animal sources have been extensively studied [ 1, 21. Among the ‘blue’ oxidases Rhus lactase from the Japanese lacquer tree recommended itself to experi- ment since it contains but four copper ions; that is, the minimal functional unit for the four electron reduction of dioxygen. In addition the moderate oxidizing strength of the copper ions (350-450 mV) made Rhus lactase more tractable than the more oxidizing fungal protein varieties. *Author to whom correspondence should be addressed. 0020-1693/84/$3.00 The four copper sites within the laccases include a single Type 1 site, with an intense absorption near 600 nm (hence the description ‘blue’), and an axial epr spectrum showing an unusually narrow parallel hyperfine coupling constant. Additionally a single Type 2 copper is present which is essentially optical- ly transparent but exhibiting a rhombic epr spectrum indicating a low symmetry ligation site. Finally the single Type 3 copper site consists of a pair of strongly antiferromagnetically coupled copper(H) ions [3, 41 associated with a strong electronic absorption band at 330 nm. It is at the Type 3 site that the binding and initial reduction of dioxygen occurs [5]. The high affinity constant (>lOs M-l) shown for the binding of hydro- gen peroxide to the oxidized Type 3 site [6], as well as the spectroscopic similarities exhibited by the bound dioxygen 2electron reduction intermediate and the Type 3 site-peroxide complex [5-81, indicated the high likelihood of similar structural forms. Other, further reduced oxygen (radical) species have also been reported and the Type 3 site copper ions suggested as the site of residence [9, 10). The preparation of a derivative of Rhus lactase depleted of Type 2 copper was reported some time ago [ 11, 121 and has been subjected to considerable study [ 12-171. The absence of Type 2 copper would allow the focus of experiments on the behavior of the Type 3 copper site, primarily as the dioxygen reduction center, manifested in the ‘blue’ oxidases. In undertaking this work, our initial experiments were concerned with defining the resting spectros- copy and oxidation state, respectively of the Type 1 and Type 3 copper ions in T2D lactase [ 171. Using a modification of the Lowry method [ 181 of protein quantitation, the extinction of the Type 1 copper at 615 nm was found to be 5620 + 570 M-’ cm-‘. This value is experimentally indistinguishable from that of the native enzyme. Anaerobic reductive titrations with Ru(NH~)~*+ and Cr(I1) aquo ions revealed that the Type 3 site copper ions are in the Cu(I1) state in the resting T2D enzyme despite the loss of their diagnostic band at 330 nm on Type 2 copper remov- al. Variations in the intensity of the 280 nm band and the reversible formation of extra epr spectral features, observed on reduction-reoxidation of T2D lactase, indicated the unreliability of the former band for 0 Elsevier Sequoia/Printed in Switzerland