S.J. Ferguson

An unusual and reversible chemical modification of soluble beef heart mitochondrial ATPase

Mitochondrial ATPase is thought to be the terminal ATP synthesising enzyme of oxidative phosphorylation [ 1 ] but little is known about its reactivity towards chemical reagents. Yet in principle, a comparison of chemical reactivity of the residues in the soluble and membrane bound enzyme could give valuable information about the location of F 1 in the membrane, and about any groups that may be essential for reconstitution of the enzyme into Fl-depleted particles [2]. Previous work has suggested that sulphydryl groups are not important in the catalytic process [3] and that modification of tyrosine residues leads to loss of activity [4]. This paper describes an unusual reaction between the ATPase from beef heart mitochondria (F 1) and 7-chloro-4-nitrobenzo-2-oxa1,3-diazole (NBD-C1) which is readily reversible and compares this modification with others brought about by more conventional chemical reagents.

Measurement by a flow dialysis technique of the steady-state proton-motive force in chromatophores from Rhodospirillum rubrum. Comparison with phosphorylation potential.

1. In the light a transmembrane electrical potential of 100 mV has been estimated to occur in chromatophores from Rhodospirillum rubrum. The potential was determined by measuring the steady-state distribution of the permeant SCN- across the chromatophore membrane using a flow dialysis technique. The potential was not observed in the dark, nor in the presence of antimycin. It was dissipated on the addition of carbonyl cyanide p-trifluoromethoxyphenylhydrazone. The potential was reduced by between 15 and 20 mV when ADP and Pi were added. Hydrolysis of ATP by the chromatophores generated a membrane potential of about 80 mV. 2. Using a flow dialysis technique light-dependent uptake of methylamine was observed only in the presence of concentrations of SCN- that were 500-fold higher than were used to measure the membrane potential. It is concluded that the pH gradient across the illuminated chromatophore membrane is insignificant except in the presence of relatively high concentrations of a permeant anion like thiocyanate. Further evidence that a negligible pH gradient was generated by the chromatophores is that addition of K+ and nigericin to illuminated chromatophores did not stimulate uptake of SCN-. 3. In the light of chromatophores established and maintained a phosphorylation potential of up to 14 kcal/mol. If a phosphorylation potential of this magnitude is to be poised against a proton-motive force that comprises solely a membrane potential of approx. 100 mV, then at least five protons must be translocated for each ATP synthesised via a chemiosmotic mechanism.

Insertion of transposon Tn5 into a structural gene of the membrane-bound nitrate reductase of Thiosphaera pantotropha results in anaerobic overexpression of periplasmic nitrate reductase activity

Chlorate-resistant mutants of the denitrifying bacterium Thiosphaera pantotropha were generated by transposon Tn5 mutagenesis. One class was deficient in membrane-bound nitrate reductase activity but retained a periplasmic nitrate reductase activity. Using transposon marker rescue it was shown that in one such mutant, M-6, the transposon was inserted in the membrane-bound nitrate reductase beta subunit structural gene (termed narH in order to be consistent with the nomenclature of the Escherichia coli major nitrate reductase operon). The translated sequence (total of 106 amino acids) from around the point of transposon insertion showed approximately 90% amino acid identity with the beta subunits of the E. coli nitrate reductases. Under anaerobic growth conditions M-6 overproduced the periplasmic nitrate reductase activity allowing anaerobic growth with nitrate as electron acceptor. A regulatory link was inferred between the presence of the membrane-bound nitrate reductase and expression of the periplasmic nitrate reductase. This is the first demonstration of full denitrification in an organism possessing only a periplasmic nitrate reductase.

Selective and reversible inhibition of the ATPase of Micrococcus denitrificans by 7-chloro-4-nitrobenzo-2-oxa-1,3 diazole

Abstract The covalent inhibitor of the beef heart mitochondrial ATPase 7-chloro-4-nitrobenzo-2-oxa-1,3 diazole inhibits the ATPase of phosphorylating particles prepared from Micrococcus denitrificans . Inhibition of both ATP synthesis and ATP hydrolysis occurs at similar rates, with a similar pH dependence, and in each case the inhibition is relieved by treatment with dithiothreitol. These results are compared with those previously obtained with the mitochondrial ATPase.

In vivo redox poising of the cyclic electron transport system of Rhodobacter capsulatus and the effects of the auxiliary oxidants, nitrate, nitrous oxide and trimethylamine N-oxide, as revealed by multiple short flash excitation

Abstract Intact cells of Rhodobacter capsulatus in the presence of myxothiazol were exposed to trains of short flashes of saturating light and the pattern of the absorbance changes due to P870, cytochrome ( c 1 + c 2 ) and the carotenoids that report on the membrane potential were monitored. Myxothiazol inhibits cyclic electron transport and therefore the extent to which electron donors and acceptors of the reaction centre are available for photochemistry is revealed. In darkened anaerobic suspensions of cells in the presence of myxothiazol, only the first two flashes in the train led to charge separation in the photosynthetic reaction centres. The results indicated that the quinone pool and quinone bound at the Q B site in the reaction centre were extensively reduced and quinone bound at Q A was partly reduced before initiation of flash excitation. Thus under these conditions, and in the absence of myxothiazol, cyclic electron transport would be restricted. In the presence of oxygen or the auxiliary oxidants trimethylamine N -oxide, NO − 3 or N 2 O, the oxidation/reduction reactions and the electrochromic absorbance changes suggested that the pool and reaction centre quinones became more oxidised. Thus, the system was poised at a potential more conducive to optimal rates of photosynthetic electron transport. By reference to experiments on the growth of Rb. capsulatus (Richardson, D.J., King, G.F., Kelly, D.J., McEwan, A.G., Ferguson, S.J. and Jackson, J.B. (1988) Arch. Microbiol. 150, 131–137), it is argued that redox poising by the auxiliary oxidants is physiologically important, especially at low light intensities. Flash train experiments reveal that over-reduction of the quinones is more severe with succinate as a carbon source than with malate and this accounts for the observation that the rate of growth on succinate is decreased more strongly at low light intensities.

Continuous monitoring of the electrical potential across energy-transducing membranes using ion-selective electrodes Application to submitochondrial particles and chromatophores

It is now widely believed that a primary result of electron transport in the energy-transducing membranes of mitochondria, chloroplasts and bacteria is the vectorial translocation of protons, leading to the generation of a transmembrane electrochemical proton gradient, the protonmotive force Ap [l] . Ap is composed of both a chemical component ApH and an electrical component A

Covalent cofactor attachment to proteins: cytochrome c biogenesis

according to the relationship :

The ATPase as an irreversible component in electron transport linked ATP synthesis

Haem (Fe-protoporphyrin IX) is a cofactor found in a wide variety of proteins. It confers diverse functions, including electron transfer, the binding and sensing of gases, and many types of catalysis. The majority of cofactors are non-covalently attached to proteins. There are, however, some proteins in which the cofactor binds covalently and one of the major protein classes characterized by covalent cofactor attachment is the c-type cytochromes. The characteristic haem-binding mode of c-type cytochromes requires the formation of two covalent bonds between two cysteine residues in the protein and the two vinyl groups of haem. Haem attachment is a complex post-translational process that, in bacteria such as Escherichia coli, occurs in the periplasmic space and involves the participation of many proteins. Unexpectedly, it has been found that the haem chaperone CcmE (cytochrome c maturation), which is an essential intermediate in the process, also binds haem covalently before transferring the haem to apocytochromes. A single covalent bond is involved and occurs between a haem vinyl group and a histidine residue of CcmE. Several in vitro and in vivo studies have provided insight into the function of this protein and into the overall process of cytochrome c biogenesis.

On the role of the essential tyrosine residue in the mitochondrial ATPase.

The reversibility of the ATPase that participates in oxidative phosphorylation is implicit within the idea that the phosphate potential (energy stored in ATP) comes into equilibrium with the respiratory chain [ 1 ] . This is the basis of an explanation of respiratory control in which ADP stimulated respiration (state 3) continues until this equilibrium is reached, whereupon respiration decreases to a controlled rate (state 4). Our purpose in this paper is to enquire whether this equilibrium view is always correct by considering the properties of the oxidative phosphorylation apparatus of ‘inside out’ phosphorylating membrane vesicles from Puracoccus denitrificans which exhibit respiratory control [2,3,4].

Thiocyanate binding to the molybdenum centre of the periplasmic nitrate reductase from Paracoccus pantotrophus.

Abstract Aurovertin has been used as a probe for the properties of an inactive derivative of bovine heart mitochondrial ATPase which was modified on just one tyrosine residue. It is suggested that this modification inhibits the enzyme by preventing a step subsequent to a conformational change produced by addition of ATP to the enzyme.