The High-Spin Heme bL Mutant Exposes Dominant Reaction Leading to the Formation of the Semiquinone Spin-Coupled to the [2Fe-2S]+ Cluster at the Qo Site of Rhodobacter capsulatus Cytochrome bc1

Cytochrome bc1 (mitochondrial complex III) catalyzes electron transfer from quinols to cytochrome c and couples this reaction with proton translocation across lipid membrane; thus, it contributes to the generation of protonmotive force used for the synthesis of ATP. The energetic efficiency of the enzyme relies on a bifurcation reaction taking place at the Qo site which upon oxidation of ubiquinol directs one electron to the Rieske 2Fe2S cluster and the other to heme bL. The molecular mechanism of this reaction remains unclear. A semiquinone spin-coupled to the reduced 2Fe2S cluster (SQo-2Fe2S) was identified as a state associated with the operation of the Qo site. To get insights into the mechanism of the formation of this state, we first constructed a mutant in which one of the histidine ligands of the iron ion of heme bLRhodobacter capsulatus cytochrome bc1 was replaced by asparagine (H198N). This converted the low-spin, low-potential heme into the high-spin, high-potential species which is unable to support enzymatic turnover. We performed a comparative analysis of redox titrations of antimycin-supplemented bacterial photosynthetic membranes containing native enzyme and the mutant. The titrations revealed that H198N failed to generate detectable amounts of SQo-2Fe2S under neither equilibrium (in dark) nor nonequilibrium (in light), whereas the native enzyme generated clearly detectable SQo-2Fe2S in light. This provided further support for the mechanism in which the back electron transfer from heme bL to a ubiquinone bound at the Qo site is mainly responsible for the formation of semiquinone trapped in the SQo-2Fe2S state in R. capusulatus cytochrome bc1.

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A Quinol Anion as Catalytic Intermediate Coupling Proton Translocation With Electron Transfer in E. coli Respiratory Complex I

Frontiers in Chemistry ◽

10.3389/fchem.2021.672969 ◽

2021 ◽

Vol 9 ◽

Author(s):

Franziska Nuber ◽

Luca Mérono ◽

Sabrina Oppermann ◽

Johannes Schimpf ◽

Daniel Wohlwend ◽

...

Keyword(s):

Electron Transfer ◽

Complex I ◽

Proton Translocation ◽

Difference Spectrum ◽

Nadh:Ubiquinone Oxidoreductase ◽

Protonmotive Force ◽

Respiratory Complex ◽

Quinone Reduction ◽

Vis Spectroscopy ◽

Respiratory Complex I

Energy-converting NADH:ubiquinone oxidoreductase, respiratory complex I, plays a major role in cellular energy metabolism. It couples NADH oxidation and quinone reduction with the translocation of protons across the membrane, thus contributing to the protonmotive force. Complex I has an overall L-shaped structure with a peripheral arm catalyzing electron transfer and a membrane arm engaged in proton translocation. Although both reactions are arranged spatially separated, they are tightly coupled by a mechanism that is not fully understood. Using redox-difference UV-vis spectroscopy, an unknown redox component was identified in Escherichia coli complex I as reported earlier. A comparison of its spectrum with those obtained for different quinone species indicates features of a quinol anion. The re-oxidation kinetics of the quinol anion intermediate is significantly slower in the D213GH variant that was previously shown to operate with disturbed quinone chemistry. Addition of the quinone-site inhibitor piericidin A led to strongly decreased absorption peaks in the difference spectrum. A hypothesis for a mechanism of proton-coupled electron transfer with the quinol anion as catalytically important intermediate in complex I is discussed.

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Cryo-EM Structures of Respiratory bc1-cbb3 type CIII2CIV Supercomplex and Electronic Communication Between the Complexes

10.1101/2020.06.27.175620 ◽

2020 ◽

Author(s):

Stefan Steimle ◽

Trevor VanEeuwen ◽

Yavuz Ozturk ◽

Hee Jong Kim ◽

Merav Braitbard ◽

...

Keyword(s):

Electron Transfer ◽

Electron Transport ◽

Rhodobacter Capsulatus ◽

Structural Features ◽

Complex Iii ◽

Transport Pathways ◽

Gram Negative ◽

Atp Production ◽

Growth Modes ◽

Respiratory Electron Transport

AbstractThe respiratory electron transport complexes convey electrons from nutrients to oxygen and generate a proton-motive force used for energy (ATP) production in cells. These enzymes are conserved among organisms, and organized as individual complexes or combined forming large super-complexes (SC). Bacterial electron transport pathways are more branched than those of mitochondria and contain multiple variants of such complexes depending on their growth modes. The Gram-negative species deploy a mitochondrial-like cytochrome bc1 (Complex III, CIII2), and may have bacteria-specific cbb3-type cytochrome c oxidases (Complex IV, CIV) in addition to, or instead of, the canonical aa3-type CIV. Electron transfer between these complexes is mediated by two different carriers: the soluble cytochrome c2 which is similar to mitochondrial cytochrome c and the membrane-anchored cytochrome cy which is unique to bacteria. Here, we report the first cryo-EM structure of a respiratory bc1-cbb3 type SC (CIII2CIV, 5.2Å resolution) and several conformers of native CIII2 (3.3Å resolution) from the Gram-negative bacterium Rhodobacter capsulatus. The SC contains all catalytic subunits and cofactors of CIII2 and CIV, as well as two extra transmembrane helices attributed to cytochrome cy and the assembly factor CcoH. Remarkably, some of the native CIII2 are structural heterodimers with different conformations of their [2Fe-2S] cluster-bearing domains. The unresolved cytochrome c domain of cy suggests that it is mobile, and it interacts with CIII2CIV differently than cytochrome c2. Distance requirements for electron transfer suggest that cytochrome cy and cytochrome c2 donate electrons to heme cp1 and heme cp2 of CIV, respectively. For the first time, the CIII2CIV architecture and its electronic connections establish the structural features of two separate respiratory electron transport pathways (membrane-confined and membrane-external) between its partners in Gram-negative bacteria.

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Novel purification of cytochrome c1 from mitochondrial Complex III. Reconstitution of antimycin-insensitive electron transfer with the iron-sulfur protein and cytochrome c1.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(18)95704-2 ◽

1985 ◽

Vol 260 (28) ◽

pp. 15075-15080

Author(s):

Y Shimomura ◽

M Nishikimi ◽

T Ozawa

Keyword(s):

Electron Transfer ◽

Complex Iii ◽

Mitochondrial Complex ◽

Iron Sulfur ◽

Cytochrome C1 ◽

Sulfur Protein ◽

Iron Sulfur Protein

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Electron transfer sensitized photolysis of 'onium salts

Canadian Journal of Chemistry ◽

10.1139/v88-055 ◽

1988 ◽

Vol 66 (2) ◽

pp. 319-324 ◽

Cited By ~ 55

Author(s):

R. J. DeVoe ◽

M. R. V. Sahyun ◽

Einhard Schmidt ◽

N. Serpone ◽

D. K. Sharma

Keyword(s):

Electron Transfer ◽

Flash Photolysis ◽

Laser Flash Photolysis ◽

Laser Flash ◽

Quantitative Model ◽

Intersystem Crossing ◽

Single Electron Transfer ◽

Onium Salts ◽

Encounter Complex ◽

Back Electron Transfer

We have studied the anthracene-sensitized photolyses of both diphenyliodonium and triphenylsulphonium salts in solution using both steady-state and laser flash photolysis techniques. Photoproducts, namely, phenylated anthracenes along with iodobenzene or diphenylsulphide, respectively, are obtained from both salts with quantum efficiencies of ca. 0.1 at 375 nm. We infer the intermediacy of diphenyliodo and triphenylsulphur radicals formed by single electron transfer from the singlet-excited anthracene. We have developed a quantitative model of this chemistry, and identify the principal sources of inefficiency as back electron transfer, which occurs at nearly the theoretically limiting rate, intersystem crossing from the initially formed sensitizer–'onium salt encounter complex, and in-cage radical recombination.

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