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Activation of respiratory Complex I from Escherichia coli studied by fluorescent probes.

ABSTRACT: Respiratory Complex I from E. coli may exist in two interconverting forms: resting (R) and active (A). The R/A transition of purified, solubilized Complex I occurring upon turnover was studied employing two different fluorescent probes, Annine 6+, and NDB-acetogenin. NADH-induced fluorescent changes of both dyes bound to solubilized Complex I from E. coli were characterized as a function of the protein:dye ratio, temperature, ubiquinone redox state and the enzyme activity. Analysis of this data combined with time-resolved optical measurements of Complex I activity and spectral changes indicated two ubiquinone-binding sites; a possibility of reduction of the tightly-bound quinone in the resting state and reduction of the loosely-bound quinone in the active state is discussed. The results also indicate that upon the activation Complex I undergoes conformational changes which can be mapped to the junction of the hydrophilic and membrane domains in the region of the assumed acetogenin-binding site.

SUBMITTER: Belevich N

PROVIDER: S-EPMC5219619 | biostudies-literature | 2017 Jan

REPOSITORIES: biostudies-literature

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Activation of respiratory Complex I from Escherichia coli studied by fluorescent probes.

Belevich Nikolai N Belevich Galina G Chen Zhiyong Z Sinha Subhash C SC Verkhovskaya Marina M

Heliyon 20170103 1

Respiratory Complex I from E. coli may exist in two interconverting forms: resting (R) and active (A). The R/A transition of purified, solubilized Complex I occurring upon turnover was studied employing two different fluorescent probes, Annine 6+, and NDB-acetogenin. NADH-induced fluorescent changes of both dyes bound to solubilized Complex I from E. coli were characterized as a function of the protein:dye ratio, temperature, ubiquinone redox state and the enzyme activity. Analysis ...[more]

PMID: 28070565

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Project description:Low concentrations (1-50mum) of ubiquinol(1) were rapidly oxidized by spheroplasts of Escherichia coli derepressed for synthesis of nitrate reductase using either nitrate or oxygen as electron acceptor. Oxidation of ubiquinol(1) drove an outward translocation of protons with a corrected -->H(+)/2e(-) stoichiometry [Scholes & Mitchell (1970) J. Bioenerg.1, 309-323] of 1.49 when nitrate was the acceptor and 2.28 when oxygen was the acceptor. Proton translocation driven by the oxidation of added ubiquinol(1) was also observed in spheroplasts from a double quinone-deficient mutant strain AN384 (ubiA(-)menA(-)), whereas a haem-deficient mutant, strain A1004a, did not oxidize ubiquinol(1). Proton translocation was not observed if either the protonophore carbonyl cyanide m-chlorophenylhydrazone or the respiratory inhibitor 2-n-heptyl-4-hydroxyquinoline N-oxide was present. When spheroplasts oxidized Diquat radical (DQ(+)) to the oxidized species (DQ(++)) with nitrate as acceptor, nitrate was reduced to nitrite according to the reaction: [Formula: see text] and nitrite was further reduced in the reaction: [Formula: see text] Nitrite reductase activity (2) was inhibited by CO, leaving nitrate reductase activity (1) unaffected. Benzyl Viologen radical (BV(+)) is able to cross the cytoplasmic membrane and is oxidized directly by nitrate reductase to the divalent cation, BV(++). In the presence of CO, this reaction consumes two protons: [Formula: see text] The consumption of these protons could not be detected by a pH electrode in the extra-cellular bulk phase of a suspension of spheroplasts unless the cytoplasmic membrane was made permeable to protons by the addition of nigericin or tetrachlorosalicylanilide. It is concluded that the protons of eqn. (3) are consumed at the cytoplasmic aspect of the cytoplasmic membrane. Diquat radical, reduced N-methylphenazonium methosulphate and its sulphonated analogue N-methylphenazonium-3-sulphonate (PMSH) and ubiquinol(1) are all oxidized by nitrate reductase via a haem-dependent, endogenous quinone-independent, 2-n-heptyl-4-hydroxyquinoline N-oxide-sensitive pathway. Approximate-->H(+)/2e(-) stoichiometries were zero with Diquat radical, an electron donor, 1.0 with reduced N-methylphenazonium methosulphate or its sulphonated analogue, both hydride donors, and 2.0 with ubiquinol(1) (QH(2)), a hydrogen donor. It is concluded that the protons appearing in the medium are derived from the reductant and the observed-->H(+)/2e(-) stoichiometries are accounted for by the following reactions occurring at the periplasmic aspect of the cytoplasmic membrane.: [Formula: see text]

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Activation of respiratory Complex I from Escherichia coli studied by fluorescent probes.

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Activation of respiratory Complex I from <i>Escherichia coli</i> studied by fluorescent probes.

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