Structural studies on copper-containing plant oxidases.

Introduction Ascorbic acid oxidase (AAO) is ;I blue multi-copper oxidase that catalyses the four-electron reduction of dioxygen to water with concomitant one-electron oxidation of tlie reducing organic substrate [ 1 1. Copper-dependent A A O is found only in higher plants 12 I. The enzyme from Cucurbitu pep0 medullosa (green zucchini) is ;I dimer of 14OOOO-M,. containing eight copper ions of three different spectroscopic forms classified ;is typeI , type-2 and type-3 according to Vinngard’s propos;il (see [ 31). ‘Hie im~n~rnoliistoclie~~iic~il lo alization of A A O in green zucchini reve;ils that AAO is distributed in all specimens examined, ubiquitously throughout vegetative and reproductive organs. At the cellular level tlie enzyme is linked with the cell \vall and cytoplasni 121. The role of ascorbate and AAO in plants in 7 i 7 ~ is still under debate. As catechols and polypheiiols are also substrates in ~ i t r o [-I], AAO might be involved in biological processes like fruit ripening. A role in a redox system, as an alternative to the mitocliondrial chain. in gro\vth promotion or in susceptibility to disease ILIS also been postulated 15 I. 1’rim;iry structures of AAO from cucumber [Sl ; i d pinipkin 17 1 have also recently been reported. 1 lie prelimin;iry three-dimensioii~il X-ray structure of the fully oxidized form of AAO from zucchini has been publishrd IS I a id the polypeptide fold and the coordination of the niononuclear blue copper site and tlie unprecedented trinuclear copper site have been described. The structure has now been refined to a resolution of 1.0 A and its detailed description and their implications tor the catalytic inechanism, electron-transfer processes, ;ind activ;ition of dioxygen have been the subject tor a further publication [ O ] . ‘I’he structural relationship to the other blue copper oxidases, laccase (EC 1.10.32) and ceruloplasmin (Ec‘ I . 1 2.3.1 ), has been demonstr;ited by amino acid sequence alignment based on the spatial structure o fAA0 from zucchini [ 101. r .