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

Berthold, Deborah A.; Siedow, James N.

doi:10.1104/pp.101.1.113

Cited by 56 publications

(36 citation statements)

References 20 publications

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“…For AOX, the large g-anisotropy (see Davydov et al,Ref. 33), the small value of the exchange coupling constant (ϪJ), the fact that the EPR signal is seen only at very low temperature, and the lack of a visible absorption at wavelengths above 320 nm (10), all point to the presence of a hydroxo (rather than oxo) bridge between the two irons. In fact, the parameters also leave open the possibility that the bridging ligand may be an aquo group.…”

Section: Discussionmentioning

confidence: 99%

“…Careful attempts made earlier to locate an EPR signal in the enzyme in whole mitochondria had been unsuccessful (35)(36)(37), as were EPR analyses of partially purified preparations (10,38).…”

Section: Discussionmentioning

confidence: 99%

“…However, continued efforts at spectroscopic detection of the enzyme were not successful. Detergent-solubilized, partial-purified preparations of this integral membrane protein yielded no EPR signal upon reduction or oxidation, and no visible absorption spectrum above 320 nm (10). Experimental evidence for the identity of the cofactor emerged when iron was shown to be required for the appearance of activity from the AOX apoprotein in whole-cell experiments with the yeast Pichia anomala (formerly Hansenula anomala) (11) and more recently for the trypanosome AOX activity in heme-deficient E. coli (12).…”

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confidence: 99%

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EPR Studies of the Mitochondrial Alternative Oxidase

Berthold

Voevodskaya

Stenmark

et al. 2002

Journal of Biological Chemistry

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The alternative oxidase (AOX) is a ubiquinol oxidase found in the mitochondrial respiratory chain of plants as well as some fungi and protists. It has been predicted to contain a coupled diiron center on the basis of a conserved sequence motif consisting of the proposed iron ligands, four glutamate and two histidine residues. However, this prediction has not been experimentally verified. Here we report the high level expression of the Arabidopsis thaliana alternative oxidase AOX1a as a maltose-binding protein fusion in Escherichia coli. Reduction and reoxidation of a sample of isolated E. coli membranes containing the alternative oxidase generated an EPR signal characteristic of a mixed-valent Fe(II)/Fe(III) binuclear iron center. The high anisotropy of the signal, the low value of the g-average tensor, and a small exchange coupling (؊J) suggest that the iron center is hydroxo-bridged. A reduced membrane preparation yielded a parallel mode EPR signal with a g-value of about 15. In AOX containing a mutation of a putative glutamate ligand of the diiron center (E222A or E273A) the EPR signals are absent. These data provide evidence for an antiferromagnetic-coupled binuclear iron center, and together with the conserved sequence motif, identify the alternative oxidase as belonging to the growing family of diiron carboxylate proteins. The alternative oxidase is the first integral membrane protein in this family, and adds a new catalytic activity (ubiquinol oxidation) to this group of enzymatically diverse proteins.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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confidence: 99%

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EPR Studies of the Mitochondrial Alternative Oxidase

Berthold

Voevodskaya

Stenmark

et al. 2002

Journal of Biological Chemistry

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“…A partially purified alternative oxidase preparation from Symplocarpus foetidus spadices also showed no EPR resonances in either the oxidized or the dithionitereduced states and had no optical absorbance above 350 nm [17]. Preliminary analysis of one S. foetidus preparation using atomic absorption spectroscopy has indicated the presence of iron (A.L.…”

Section: Introductionmentioning

confidence: 99%

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

1995

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The cyanide-resistant, alternative oxidase of plant mitochondria catalyzes the four-electron reduction of oxygen to water, but the nature of the catalytic center associated with this oxidase has yet to be elucidated. We have identified conserved amino acids, including two copies of the iron-binding motif GIu-X-X-His, in the carboxy-terminal hydrophilic domain of the alternative oxidase that suggest the presence of a hydroxo-bridged binuclear iron center, analogous to that found in the enzyme methane monooxygenase. Using the known three-dimensional structures of other binuclear iron proteins, we have developed a structural model for the proposed catalytic site of the alternative oxidase based on these amino acid sequence similarities.

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“…The current model of the AOX, supported by mutagenesis studies, predicts a monotopic integral membrane protein (2,(13)(14)(15) associating with one leaflet of the lipid bilayer. Although analyses of yeast and trypanosomal enzymes have established that iron is required for activity (16,17), early investigations of either mitochondria or partially purified protein failed to reveal spectroscopic signatures of its active site (18,19). The first spectroscopic evidence for iron involvement was provided by Berthold et al (20) (20) were not successful.…”

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Three Redox States of Trypanosoma brucei Alternative Oxidase Identified by Infrared Spectroscopy and Electrochemistry

Maréchal

Kido

Kita

et al. 2009

Journal of Biological Chemistry

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Electrochemistry coupled with Fourier transform infrared (IR) spectroscopy was used to investigate the redox properties of recombinant alternative ubiquinol oxidase from Trypanosoma brucei, the organism responsible for African sleeping sickness.Stepwise reduction of the fully oxidized resting state of recombinant alternative ubiquinol oxidase revealed two distinct IR redox difference spectra. The first of these, signal 1, titrates in the reductive direction as an n ‫؍‬ 2 Nernstian component with an apparent midpoint potential of 80 mV at pH 7.0. However, reoxidation of signal 1 in the same potential range under anaerobic conditions did not occur and only began with potentials in excess of 500 mV. Reoxidation by introduction of oxygen was also unsuccessful. Signal 1 contained clear features that can be assigned to protonation of at least one carboxylate group, further perturbations of carboxylic and histidine residues, bound ubiquinone, and a negative band at 1554 cm ؊1 that might arise from a radical in the fully oxidized protein. A second distinct IR redox difference spectrum, signal 2, appeared more slowly once signal 1 had been reduced. This component could be reoxidized with potentials above 100 mV. In addition, when both signals 1 and 2 were reduced, introduction of oxygen caused rapid oxidation of both components. These data are interpreted in terms of the possible active site structure and mechanism of oxygen reduction to water.Mitochondria from many higher plants possess, in addition to the conventional cytochrome c oxidase, a second terminal oxidase that oxidizes ubiquinol (1-3). In thermogenic plants this alternative oxidase (AOX) 6 plays a key role in the release of heat for pollination purposes or for maintaining a warm environment within the flower at low ambient temperatures. In nonthermogenic plants its function is still under debate; proposed roles include maintaining tricarboxylic acid cycle turnover under high cytosolic phosphorylation potentials, defense against oxidative stress, and growth rate and energy charge homeostasis (4). AOX is also found in species of fungi, green algae, bacteria, and protozoa (5) and, more recently, in mollusks, nematodes, and chordates (6). Of particular importance, however, is its presence in pathogenic protozoa such as the blood parasite Trypanosoma brucei (7) and the intestinal parasite Cryptosporidium parvum (8, 9). T. brucei is a parasite that causes African sleeping sickness in humans and Nagana in livestock and is transmitted by the tsetse fly (7). The bloodstream forms of T. brucei appear to depend solely on its alternative oxidase (TAO) for respiration. Because the protein is absent from the mammalian host, TAO is an attractive and important chemotherapeutic target for African trypanosomiasis (7-10). In this respect it is interesting to note that ascofuranone, isolated from the pathogenic fungus Ascochyta visiae, specifically and potently inhibits the quinol oxidase activity of TAO (11) and rapidly kills the parasites. In addition, the chemotherapeutic eff...

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

Cited by 56 publications

References 20 publications

EPR Studies of the Mitochondrial Alternative Oxidase

EPR Studies of the Mitochondrial Alternative Oxidase

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

Three Redox States of Trypanosoma brucei Alternative Oxidase Identified by Infrared Spectroscopy and Electrochemistry

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