Dataset Information

Kinetic analysis of the mitochondrial quinol-oxidizing enzymes during development of thermogenesis in Arum maculatum L.

ABSTRACT: The dependence of the rate of oxygen uptake upon the ubiquinone (Q)-pool reduction level in mitochondria isolated during the development of thermogenesis of Arum maculatum spadices has been investigated. At the alpha-stage of development, the respiratory rate was linearly dependent upon the reduction level of the Q-pool (Qr) both under state-3 and -4 conditions. Progression through the beta/gamma to the delta-stage resulted in a non-linear dependence of the state-4 rate on Qr. In the delta-stage of development, both state-3 and -4 respiratory rates were linearly dependent upon Qr due to a shift in the engagement of the alternative oxidase to lower levels of Qr. Western blot analysis revealed that increased alternative oxidase activity could be correlated with expression of a 35 kDa protein. Respiratory control was only observed with mitochondria in the alpha-stage of development. At the beta/gamma-stage of development, the addition of ADP resulted in a significant oxidation of the Q-pool which was accompanied by a decrease in the respiratory rate. This was due either to decreased contribution of the alternative pathway to the overall respiratory rate under state 3 or by deactivation of succinate dehydrogenase activity by ADP. Cold-storage of the spadices at the beta-stage of development led to increased activity of both the cytochrome pathway and succinate dehydrogenase, without any change in alternative oxidase activity. Results are discussed in terms of how changes in the activation level of the alternative oxidase and succinate dehydrogenase influence the activity and engagement of the quinol-oxidizing pathways during the development of thermogenesis in A. maculatum.

SUBMITTER: Leach GR

PROVIDER: S-EPMC1217480 | biostudies-other | 1996 Jul

REPOSITORIES: biostudies-other

ACCESS DATA

Similar Datasets

Project description:Sulfite oxidizing enzymes are essential mononuclear molybdenum (Mo) proteins involved in sulfur metabolism of animals, plants and bacteria. There are three such enzymes presently known: (1) sulfite oxidase (SO) in animals, (2) SO in plants, and (3) sulfite dehydrogenase (SDH) in bacteria. X-ray crystal structures of enzymes from all three sources (chicken SO, Arabidopsis thaliana SO, and Starkeya novella SDH) show nearly identical square pyramidal coordination around the Mo atom, even though the overall structures of the proteins and the presence of additional cofactors vary. This structural information provides a molecular basis for studying the role of specific amino acids in catalysis. Animal SO catalyzes the final step in the degradation of sulfur-containing amino acids and is critical in detoxifying excess sulfite. Human SO deficiency is a fatal genetic disorder that leads to early death, and impaired SO activity is implicated in sulfite neurotoxicity. Animal SO and bacterial SDH contain both Mo and heme domains, whereas plant SO only has the Mo domain. Intraprotein electron transfer (IET) between the Mo and Fe centers in animal SO and bacterial SDH is a key step in the catalysis, which can be studied by laser flash photolysis in the presence of deazariboflavin. IET studies on animal SO and bacterial SDH clearly demonstrate the similarities and differences between these two types of sulfite oxidizing enzymes. Conformational change is involved in the IET of animal SO, in which electrostatic interactions may play a major role in guiding the docking of the heme domain to the Mo domain prior to electron transfer. In contrast, IET measurements for SDH demonstrate that IET occurs directly through the protein medium, which is distinctly different from that in animal SO. Point mutations in human SO can result in significantly impaired IET or no IET, thus rationalizing their fatal effects. The recent developments in our understanding of sulfite oxidizing enzyme mechanisms that are driven by a combination of molecular biology, rapid kinetics, pulsed electron paramagnetic resonance (EPR), and computational techniques are the subject of this review.

Project description:In this paper we present theoretical calculations on model biomimetic systems for quinol oxidation. In these model systems, an excited-state [Ru(bpy)(2)(pbim)](+) complex (bpy = 2,2'-dipyridyl, pbim = 2-(2-pyridyl)benzimidazolate) oxidizes a ubiquinol or plastoquinol analogue in acetonitrile. The charge transfer reaction occurs via a proton-coupled electron transfer (PCET) mechanism, in which an electron is transferred from the quinol to the Ru and a proton is transferred from the quinol to the pbim(-) ligand. The experimentally measured average kinetic isotope effects (KIEs) at 296 K are 1.87 and 3.45 for the ubiquinol and plastoquinol analogues, respectively, and the KIE decreases with temperature for plastoquinol but increases with temperature for ubiquinol. The present calculations provide a possible explanation for the differences in magnitudes and temperature dependences of the KIEs for the two systems and, in particular, an explanation for the unusual inverse temperature dependence of the KIE for the ubiquinol analogue. These calculations are based on a general theoretical formulation for PCET reactions that includes quantum mechanical effects of the electrons and transferring proton, as well as the solvent reorganization and proton donor-acceptor motion. The physical properties of the system that enable the inverse temperature dependence of the KIE are a stiff hydrogen bond, which corresponds to a high-frequency proton donor-acceptor motion, and small inner-sphere and solvent reorganization energies. The inverse temperature dependence of the KIE may be observed if the 0/0 pair of reactant/product vibronic states is in the inverted Marcus region, while the 0/1 pair of reactant/product vibronic states is in the normal Marcus region and is the dominant contributor to the overall rate. In this case, the free energy barrier for the dominant transition is lower for deuterium than for hydrogen because of the smaller splittings between the vibronic energy levels for deuterium, and the KIE increases with increasing temperature. The temperature dependence of the KIE is found to be very sensitive to the interplay among the driving force, the reorganization energy, and the vibronic coupling in this regime.

Dataset Information

Kinetic analysis of the mitochondrial quinol-oxidizing enzymes during development of thermogenesis in Arum maculatum L.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets