Cynthia Hou

Insights into the respiratory electron transfer pathway from the structure of nitrate reductase A

The facultative anaerobe Escherichia coli is able to assemble specific respiratory chains by synthesis of appropriate dehydrogenases and reductases in response to the availability of specific substrates. Under anaerobic conditions in the presence of nitrate, E. coli synthesizes the cytoplasmic membrane-bound quinol-nitrate oxidoreductase (nitrate reductase A; NarGHI), which reduces nitrate to nitrite and forms part of a redox loop generating a proton-motive force. We present here the crystal structure of NarGHI at a resolution of 1.9 Å. The NarGHI structure identifies the number, coordination scheme and environment of the redox-active prosthetic groups, a unique coordination of the molybdenum atom, the first structural evidence for the role of an open bicyclic form of the molybdo-bis(molybdopterin guanine dinucleotide) (Mo-bisMGD) cofactor in the catalytic mechanism and a novel fold of the membrane anchor subunit. Our findings provide fundamental molecular details for understanding the mechanism of proton-motive force generation by a redox loop.

Subunit composition, function, and spatial arrangement in the Ca2+-and Mg2+-activated adenosine triphosphatases of Escherichia coli and Salmonella typhimurium.

Abstract The Ca2+- and Mg2+-activated ATPases of Escherichia coli NRC 482 and Salmonella typhimurium LT2 were purified to homogeneity. Both enzymes consisted of five polypeptides (α-ϵ). The molecular weights of the α, β, and ϵ polypeptides were 56,800, 51,800 and 13,200 for both enzymes. The molecular weights of the γ and δ polypeptides of the E. coli and S. typhimurium ATPases were 32,000 and 20,700, and 30,900 and 21,500, respectively. In both ATPases the stoichiometry of the subunits was α3β3γδϵ as determined with the 14C-labeled enzymes. The ATPases of either organism reacted with equal effectiveness with ATPase-deficient particles of the other organism to reconstitute energy-dependent transhydrogenase activity. Treatment of the homogeneous ATPases of both organisms with TPCK-trypsin stimulated ATPase activity but resulted in destruction of coupling factor activity. Trypsin treatment completely digested the δ and ϵ polypeptides, and removed up to 70% of the γ polypeptide. In the presence of the bifunctional cross-linking reagent dithiobis(succinimidyl propionate) ATPase activity was lost and cross-linking of α to β polypeptides occurred. Crosslinking of α to α or β to β polypeptides was not detected. The function of the individual polypeptides of the ATPase is discussed and a model for their spatial arrangement in the enzyme is presented.

Organization of proteins in the native and reformed outer membrane of Escherichia coli

1. 1. The proteins of the outer membrane of Escherichia coli were characterized by polyacrylamide gel electrophoresis and their molecular weights determined. 2. 2. A buffer system is described which permits reproducible resolution of three proteins which do not resolve on polyacrylamide gel electrophoresis using conventional phosphate-sodium dodecyl sulfate buffer systems. 3. 3. Pronase digestion of spheroplast membranes, Triton-treated cell envelopes and isolated outer membranes of E. coli showed that only two major proteins were accessible to the enzyme in spheroplast membranes, while Triton treatment revealed several others to pronase attack. Several other proteins were not attacked by pronase under the conditions employed. An asymmetric arrangement of proteins within the membrane is suggested. 4. 4. Pronase digestion of reformed outer membrane gave a result similar to that obtained for the original outer membrane preparation. It is suggested that reaggregation of the components of the solubilized outer membrane has occurred in a specific manner with the proteins organized as in the native membrane.

Function of energy-dependent transhydrogenase in Escherichia coli

Abstract The activity of the energy-dependent transhydrogenase of membrane particles of E. coli varied markedly with growth conditions The activity of the enzyme was not related to the efficiency of oxidative phosphorylation. The enzyme was not subject to catabolite repression but was repressed by mixtures of amino acids. It is suggested that the transhydrogenase has a role in generating NADPH for biosynthesis.

Effect of removal or modification of subunit polypeptides on the coupling factor and hydrolytic activities of the Ca2+ and Mg2+-activated adenosine triphosphatase of Escherichia coli

Abstract The effect of removal or modification of the polypeptide subunits (α, β γ, δ, and ϵ) of the Ca 2+ and Mg 2+ -activated ATPase of Escherichia coli was investigated. Removal of the δ-polypeptide, although giving some decrease in ATPase activity, resulted in complete loss of coupling activity, where coupling activity was measured by the restoration of the energy-dependent transhydrogenase activity of ATPase-stripped respiratory particles. Modification of the γ-polypeptide, as found in the ATPase of an energy transfer coupling mutant ( etc-15 ), resulted in diminution of the ATPase and coupling activities. The diminished coupling activity could be overcome by using more of the enzyme which suggested that this enzyme may not be able to bind to the membrane as firmly as the enzyme from the wild type.

Crosslinking and radiation inactivation analysis of the subunit structure of the pyridine nucleotide transhydrogenase of Escherichia coli

The pyridine nucleotide transhydrogenase of Escherichia coli consists of two types of subunit (alpha: Mr 53,906; beta: Mr 48,667). The purified and membrane-bound enzymes were crosslinked with a series of bifunctional crosslinking agents and by catalyzing the formation of inter-chain disulfides in the presence of cupric 1,10-phenanthrolinate. Crosslinked dimers alpha 2, alpha beta and beta 2, and the trimer alpha 2 beta were obtained. A small amount of tetramer, probably alpha 2 beta 2, was also formed. Radiation inactivation was used to determine the molecular size of the transhydrogenase. The radiation inactivation size (217,000) and chemical crosslinking are consistent with the structure (Mr 205,146) being the oligomer that is responsible for biological activity.

Reconstitution of energy-dependent transhydrogenase in ATPase-negative mutants of Escherichia coli

Abstract The aerobic-driven and ATP-driven energy-dependent transhydrogenase activities of membrane particles from two different Ca2+, Mg2+-activated ATPase-negative mutants of E. coli were examined. The activities were low or absent in one of the mutants (DL-54). Reconstitution of the aerobic-driven reaction could be obtained by addition to particles from this mutant of DCCD or of a coupling factor prepared from the parent strain. The coupling factor also restored the ATP-driven reaction. In the other mutant (N144) the aerobic-driven activity was unimpaired, and was not affected by DCCD or by the coupling factor. The difference between the two mutants could be rationalized if the coupling factor ATPase had both a stabilizing and an enzymic function.

Purification and characterization of the inactive Ca2+, Mg2+-activated adenosine triphosphatase of the unc A− mutant Escherichia coli AN120

Abstract The inactive Ca2+, Mg2+-activated ATPase of the une A− mutant Escherichia coli AN120 and the active enzyme of the normal strain AN180 were purified to homogeneity. Both enzymes contained five different subunits (α-ϵ) with the same molecular weights and stoichiometry. Both enzymes gave identical cross-linked products with the bifunctional reagent dithiobis(succinimidyl propionate), suggesting that the subunits were arranged in the same way in the two enzymes. Treatment of the enzyme of AN120 with Natosylphenylalanine chloromethyl ketone-trypsin did not reveal latent ATPase activity. The ATPase of AN120 bound normally to ATPase-depleted membranes of AN180 or AN120 to reconstitute aerobic-driven energy-dependent transhydrogenase activity. The enzymes from both AN120 and AN180 contained one to two molecules of both ATP and ADP per molecule of ATPase. Each enzyme could also bind one molecule of exogenous [3H]ADP per molecule of ATPase. The dissociation constants of the [3H]ADP-ATPase complexes formed by the enzymes from AN120 and AN180 were 3.55 and 9.3 μ m , respectively. Both enzymes reacted with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole with the substitution of a tyrosine residue at the active site. It is concluded that the loss of hydrolytic activity in the ATPase of AN120 is due to an alteration at the active site which is reflected in nucleotide binding but may involve a catalytically active amino acid.

Reduced nicotinamide adenine dinucleotide oxidation in Escherichia coli particles. I. Properties and cleavage of the electron transport chain.

Abstract A small particle fraction was isolated from sonic extracts of Escherichia coli . This fraction contained flavin and cytochromes b 1 and o . NADH was oxidized but not succinate, formate, pyruvate, or NADPH. Low amounts of coenzyme Q 8 (0.33 μmole/ gm protein) were present. The lipoquinone appeared to be on a minor pathway of electron transport as iso-octane extraction had little effect on the overall rate of NADH oxidation. NADH oxidation was inhibited notably by cyanide, 2-heptyl-4hydroxyquinoline N -oxide, dicumarol, o -phenanthroline, and 2,2′-dipyridyl. The inhibition by 2,2′-dipyridyl was competitive with substrate. The small particle fraction was dissociated by deoxycholate-ammonium sulfate into a cytochrome-containing particle and two NADH-menadione reductases. One of these enzymes also contained a soluble NADH oxidase.

Effect of disulfide cross-linking between α and δ subunits on the properties of the F1 adenosine triphosphatase of Escherichia coli

Abstract Under very mild oxidizing conditions the δ subunit of the F 1 -ATPase of Escherichia coli can be crosslinked by a disulfide linkage to one of the α subunits of the enzyme. The cross-linked ATPase resembles the native enzyme in the following properties: (1) specific activity; (2) activation by lauryldimethylamine N -oxide (LDAO); (3) binding of aurovertin D and ADP; (4) cross-linking products with 3,3′-dithiobis(succinimidyl propionate); (5) binding to ATPase-stripped everted membrane vesicles and the N , N ′-dicyclohexylcarbodiimide sensitivity of the rebound enzyme. However, the rebound crosslinked ATPase differed from the native enzyme in lacking the ability to restore NADH oxidation - and ATP hydrolysis-dependent quenching of the fluorescence of quinacrine to ATPase-stripped membrane vesicles. It is proposed that the δ subunit is involved in the proton pathway of the ATPase, and that this pathway is affected in the αδ-cross-linked enzyme. The mechanism for activation of the ATPase by LDAO was examined. Evidence against the proposal of Lotscher, H.-R., De Jong, C. and Capaldi, R.A. (Biochemistry (1984) 23, 4140–4143) that activation involves displacement of the e subunit from an active site on a β subunit was obtained.