Partial Purification and Characterization of the Quinol Oxidase Activity of Arum maculatum Mitochondria

Abstract

The menadiol oxidase activity of Arum maculatum mitochondria has been solubilized and fractionated. A preparation has been obtained which has an increased specific activity and a greatly decreased polypeptide composition when compared to the mitochondria. This preparation retains normal inhibitor sensitivities in that the oxidation of menadiol remains insensitive to cyanide and is inhibited by aromatic hydroxamates. Metal analyses of the preparation showed that only iron was closely correlated with the oxidase activity. No unusual lipid components were detected in the preparation. The results are discussed in relation to chemical quinol oxidation mechanisms and to several recent hypotheses concerning the nature of the higher plant alternative oxidase.The phenomenon of thermogenesis in plants has been documented for over 200 years but it was not until the 1930s that van Herk (28) was able to ascribe the process to the presence of a cyanide-insensitive respiratory pathway which subsequently became known as the alternative pathway. The molecular nature of this pathway has however remained obscure, because of the apparent lack of any distinguishing spectroscopic features of the components involved. The diverse suggestions of its possible nature have been the subject of several relatively recent reviews (1,10,13,23 quinol oxidase preparation. These results are discussed in relation to the above-mentioned suggestions as to the nature of the oxidase.MATERIALS AND METHODS Preparations. Mitochondria were prepared from the unopened spadices ofArum maculatum by a standard procedure (3). They were either used immediately or stored in liquid N2 until required. Protein content of the mitochondria was estimated from the amount of Cyt b present and assuming 0.4 nmol Cyt b/mg protein. The Cyt b was estimated from a dithionite-reduced minus oxidized difference spectrum and with an extinction coefficient at 560 nm of 20 mm-' cm-'.Menadiol was prepared from the quinone by a method described previously (16). The final solid was a light purple color and was stable for several months under N2. A stock solution of 50 mm in 96% ethanol with 10 mM HCI was used. This solution was colorless and stable for several weeks at -20°C. It was discarded as soon as any coloration was noted.Quinol Oxidase Assay. Activity was measured under dark conditions with a Clark-type 02 electrode in a buffer containing 50 mM Mes, 2 mm EDTA, and 1 mm KCN at 25°C and pH 6.0; 0.5 mM menadiol was added and the basal autoxidation rate was noted. The nonenzymic rate was always in the range of 5 to 10 nmol 02 consumed/ml min. The reaction was then started by addition of an appropriate amount of enzyme. That the stimulation observed was caused by quinol oxidase was confirmed by inhibition with 1 mm SHAM.2 Rates are pseudo-first order with quinol and hence the reaction profiles are curved. Rates were estimated from the initial part of these curves. Units of oxidase activity are defined as nmol 02 consumed/min at 25C and 0.5 mM menadiol.Spectra.

Partial Purification of the Cyanide-Resistant Alternative Oxidase of Skunk Cabbage (Symplocarpus foetidus) Mitochondria

“…Huq and Palmer (1978) reported an absorbance in this region in their partially purified alternative oxidase preparation from Arum maculatum, which they attributed to carotenoid. The later report of the partial purification of the Arum alternative oxidase by Bonner et al (1986) also mentions the presence of carotenoid. An analysis of the protein profile of each purification step using SDS-PAGE is shown in Figure 2.…”

“…Another previously unrecognized difficulty in the purification of the aroid alternative oxidase was the presence of a highly active sulfhydryl protease activity. This protease may also be present in the spadix tissue of other aroids, and may account for.the observation of Bonner et al (1986) that the alternative oxidase was "extraordinarily heat-labile after solubilization." It was found that isolation of spadix mitochondria in the presence of E-64 inhibited this protease activity without any deleterious effect on the alternative oxidase activity.…”

“…There are a number of reasons why purification of the alternative oxidase has been difficult to achieve in spite of the efforts of several laboratories (Huqand Palmer, 1978;Bonner and Rich, 1983;Bonner et al, 1986;McIntosh, 1986, 1987). First, ubiquinol and ubiquinol analogs donate electrons to the alternative oxidase of nonaroid spadix mitochondria only at very low rates.…”

The active site of the cyanide‐resistant oxidase from plant mitochondria contains a binuclear iron center

“…E-mail: jsiedow@acpub.duke.edu coupled transition metal center, by analogy with other known water-forming oxidases [10]. Previous metal analyses of partially purified alternative oxidase preparations have been inconclusive; Fe, Cu and Mn have each been reported in varying amounts [11][12][13]. Among the alternative oxidase amino acid sequences, no motifs common to metal binding sites reported for other proteins have been identified to date [1,3,7].…”

Respiration in Cells and Mitochondria of Male-Fertile and Male-Fertile and Male-Sterile Nicotiana spp.

“…Lambers (20) suggested that the alternative pathway functions as an overflow mechanism when the cytochrome pathway is saturated. An additional terminal oxidase responsible for the activity of this alternative respiratory pathway has been identified (6,11,12).…”