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Both plant and fungal mitochondria have cyanide-resistant alternative oxidases that use reductant from the mitochondrial ubiquinone pool to reduce oxygen to water in a reaction that conserves no energy for ATP synthesis. The dimeric plant alternative oxidase is relatively inactive when its subunits are linked by a disulfide bond. When this bond is reduced, the enzyme can then be stimulated by its activators, alpha-keto acids. A Cys in the N-terminal section of the protein is responsible for both of these features. We examined the alternative oxidases in mitochondria isolated from two fungi Neurospora crassa and Pichia stipitis for dimeric structure, ability to form an intermolecular disulfide, and sensitivity to alpha-keto acids. Neither of the two fungal alternative oxidases could be covalently linked by diamide, which induces disulfide bond formation between nearby Cys residues, nor could they be cross-linked by a Lys-specific reagent or glutaraldehyde at concentrations which cross-link the plant alternative oxidase dimer completely. Alternative oxidase activity in fungal mitochondria was not stimulated by the alpha-keto acids pyruvate and glyoxylate. Pyruvate did stimulate activity when succinate was the respiratory substrate, but this was not a direct effect on the alternative oxidase. In contrast, added GMP was a strong activator of fungal alternative oxidase activity. Analysis of plant and fungal alternative oxidase protein sequences revealed a unique domain of about 40 amino acids surrounding the regulatory Cys in the plant sequences that is not present in the fungal sequences. This domain may be where dimerization of the plant enzymes occurs. In contrast to plant enzymes, the fungal alternative oxidases studied here are monomeric and their activities are independent of alpha-keto acids., (Copyright 2000 Academic Press.)

URF13 is a mitochondrially encoded protein in the inner mitochondrial membrane of maize (Zea mays L.) carrying the cms-T cytoplasm. This protein is responsible for Texas-type cytoplasmic sterility and is a ligand-gated, pore-forming receptor for the pathotoxins of fungal pathogens Bipolaris maydis race T and Phyllosticta maydis. URF13 contains three transmembrane alpha-helices, with amphipathic helices II and III likely involved in pore formation, and is present as oligomers in cms-T maize mitochondria and when expressed in Escherichia coli cells. To study tertiary and quaternary structures of URF13 oligomers, we employed combinations of site-directed mutagenesis and chemical cross-linking. We introduced Cys residues individually into consecutive positions 78-82, believed to be in helix III. We expressed these proteins in E. coli cells and tested for cross-linking through disulfide bond formation or by using Cys-Cys cross-linkers. URF13-R79C, URF13-R81C, and URF13-T82C were cross-linked using Cys-Cys-specific cross-linkers, as were double mutants URF13-C27R/R79C, URF13-C27R/R81C, and URF13-C27R/T82C, indicating that the cross-linking was between introduced Cys residues on adjacent URF13 molecules. Disulfide bond formation, induced by diamide, was seen only in URF13-T82C and URF13-C27R/T82C, indicating that Cys residues introduced into position 82 are closely juxtaposed in the oligomers. Based on these observations, we modified the models for the secondary structure of URF13 and the tertiary structure of the URF13 oligomers. Sequential cross-linking of URF13-R81C oligomers with bismaleimidohexane (Cys-Cys cross-linker) and N,N'-dicyclohexylcarbodiimide (Lys-Asp/Glu cross-linker) suggests that URF13 oligomers consist of an even number of monomers.

The redox poise of the ubiquinone pool during electron transfer in isolated soybean mitochondria has been compared using two different procedures: the rapid organic extraction of ubiquinone followed by quantification of the oxidized and reduced forms using high-pressure liquid chromatography and an electrochemical technique that measures ubiquinone reduction voltametrically. The goal of these studies was to rigorously test the use of the voltametric technique to monitor the redox status of ubiquinone during the course of mitochondrial electron transfer. The linear relationship between the two methods confirms the reliability of the data obtained with the voltametric technique; however, redox inactive pools of ubiquinone were detected with the HPLC technique. We also quantified the absolute amounts of the ubiquinone homologues ubiquinone-9 and ubiquinone-10 in mitochondria isolated from different soybean tissues and compared their behavior during electron transfer in the presence and absence of pyruvate, an allosteric effector of the cyanide-resistant electron transfer pathway. Both homologues belong to a common redox-active pool in the inner mitochondrial membrane. The results indicate that ubiquinone can be a limiting component of electron transfer through the cyanide-resistant pathway, particularly in roots where its concentration is much lower than in cotyledons.

An affinity-purified polyclonal antibody against soybean seed lipoxygenase-2 was prepared and used to characterize the immunological relatedness of lipoxygenase isozymes 1 and 2 and lipoxygenases from soybean seedling roots, hypocotyls, leaves, and cotyledons. All soybean lipoxygenases tested cross-reacted with the anti-lipoxygenase-2. Cross-reactivity of seed-derived lipoxygenases was evidenced by formation of a line of identity in double-diffusion tests, by positive results on an immunoblot, and by antibody precipitation of enzyme activity. Levels of anti-lipoxygenase-2, which inhibited lipoxygenase-2 activity, had no effect on lipoxygenase-1 activity. Root, hypocotyl, and leaf lipoxygenases did not form precipitation lines in double-diffusion tests but the anti-lipoxygenase-2 did inhibit and precipitate lipoxygenase activity from these sources as well as cross-react on immunoblots. All the cross-reactive lipoxygenases examined were found to have the same apparent molecular weight. Lipoxygenase activity found in soybean seedling roots, hypocotyls, leaves, and cotyledons is associated with proteins which are all immunologically related to the seed-derived enzymes.