A Cytochrome f/b6 Complex of Five Polypeptides with Plastoquinol‐Plastocyanin‐Oxidoreductase Activity from Spinach Chloroplasts

Hurt, Eduard C.; Hauska, Günter

doi:10.1111/j.1432-1033.1981.tb06379.x

Cited by 396 publications

(40 citation statements)

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“…Thylakoid membranes were isolated from spinach as described in ref. 15. Cyt b 6 f was isolated according to the procedure described in SI Materials and Methods.…”

Section: Methodsmentioning

confidence: 99%

Metastable radical state, nonreactive with oxygen, is inherent to catalysis by respiratory and photosynthetic cytochromes bc ₁ / b ₆ f

Sarewicz

Bujnowicz

Bhaduri

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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Oxygenic respiration and photosynthesis based on quinone redox reactions face a danger of wasteful energy dissipation by diversion of the productive electron transfer pathway through the generation of reactive oxygen species (ROS). Nevertheless, the widespread quinone oxido-reductases from the cytochrome bc family limit the amounts of released ROS to a low, perhaps just signaling, level through an as-yet-unknown mechanism. Here, we propose that a metastable radical state, nonreactive with oxygen, safely holds electrons at a local energetic minimum during the oxidation of plastohydroquinone catalyzed by the chloroplast cytochrome b 6 f. This intermediate state is formed by interaction of a radical with a metal cofactor of a catalytic site. Modulation of its energy level on the energy landscape in photosynthetic vs. respiratory enzymes provides a possible mechanism to adjust electron transfer rates for efficient catalysis under different oxygen tensions.cytochrome b 6 f | reactive oxygen species | semiquinone | electron paramagnetic resonance | electron transport P hotosynthetic and respiratory cytochromes bc 1 /b 6 f (Fig. 1A) generate a proton-motive force (pmf) that powers cellular metabolism by using the Gibbs free energy difference (ΔG) between hydroquinone (QH 2 ) derivatives (Fig. 1B) and oxidized soluble electron transfer proteins (e.g., cytochrome c or plastocyanin) (1, 2). To increase the efficiency of this process, which is critical for the yield of the generated pmf, one part of the enzyme recirculates electrons to the quinone pool in the membrane (Q pool), whereas the second part steers the electrons to the cytochrome c pool, powering the electron recirculation (Fig. 1C). This mechanism, which is best established for the cytochrome bc 1 (cyt bc 1 ) (3, 4), with supporting data for the cytochrome b 6 f (cyt b 6 f) (5), discussed in ref. 2, is based on bifurcation of the route for two electrons released upon oxidation of QH 2 at one of the catalytic sites-the Q p site, (Q p ), (Fig. 1D) (3-5). A model for the energetics of this reaction assumes that one electron derived from the two-electron QH 2 donor is transferred, through the high-potential cofactor chain ("steering part" in Fig. 1C) to plastocyanin or cytochrome c, whereas the second electron is transferred across the membrane through low-potential cofactors ("recirculation" part in Fig. 1C).The electronic bifurcation process requires formation of a short-lived and reducing redox intermediate-ubisemiquinone (USQ) or plastosemiquinone (PSQ) (4, 6, 7). However, such an intermediate in an oxygenic environment would readily reduce oxygen to form superoxide radical, (O 2 − ), compromising the efficiency of energy conservation (8). Even in cyt b 6 f where the level of superoxide production through this pathway is at least an order of magnitude greater than that from yeast cyt bc 1 , the branching ratio for electron transfer to O 2 forming O 2 − is only 1-2% of the total flux (6). The low absolute level of O 2 − production in native proteins implies the e...

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“…Thylakoid membranes were isolated from spinach as described in ref. 15. Cyt b 6 f was isolated according to the procedure described in SI Materials and Methods.…”

Section: Methodsmentioning

confidence: 99%

Metastable radical state, nonreactive with oxygen, is inherent to catalysis by respiratory and photosynthetic cytochromes bc ₁ / b ₆ f

Sarewicz

Bujnowicz

Bhaduri

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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“…Since the first isolation of an active b 6 f complex from spinach (Spinacia oleracea) chloroplasts (Hurt and Hauska, 1981), the purification strategy has been continuously improved and used for many photoautotrophic organisms ranging from cyanobacteria to green algae. Considerable progress resulted from the introduction of molecular genetic tag systems for the protein complex isolation, which reduced the purification time and with it the exposure time to detergents and other disintegrating components.…”

Section: Isolation Of Stable Dimeric B 6 F Complexes Indicates Structmentioning

confidence: 99%

“…Activity of isolated complexes was assayed according to (Hurt and Hauska, 1981;Metzger et al, 1997) using 50 mM plastochinol as electron donor, 0.15 mM b 6 f complex and 20 mM cytochrome c as electron acceptor. The redox state of cytochrome c was monitored by spectrophotometry at 550 nm.…”

Section: Construction Of a Dpetp Mutantmentioning

confidence: 99%

Functional Characterization of the Small Regulatory Subunit PetP from the Cytochrome b₆f Complex in Thermosynechococcus elongatus

Rexroth

Veit

et al. 2014

Plant Cell

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The cyanobacterial cytochrome b 6 f complex is central for the coordination of photosynthetic and respiratory electron transport and also for the balance between linear and cyclic electron transport. The development of a purification strategy for a highly active dimeric b 6 f complex from the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1 enabled characterization of the structural and functional role of the small subunit PetP in this complex. Moreover, the efficient transformability of this strain allowed the generation of a DpetP mutant. Analysis on the whole-cell level by growth curves, photosystem II light saturation curves, and P700 + reduction kinetics indicate a strong decrease in the linear electron transport in the mutant strain versus the wild type, while the cyclic electron transport via photosystem I and cytochrome b 6 f is largely unaffected. This reduction in linear electron transport is accompanied by a strongly decreased stability and activity of the isolated DpetP complex in comparison with the dimeric wild-type complex, which binds two PetP subunits. The distinct behavior of linear and cyclic electron transport may suggest the presence of two distinguishable pools of cytochrome b 6 f complexes with different functions that might be correlated with supercomplex formation.

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“…Cyt b 6 f complex is also a heme-containing protein complex and shows peroxidase activity, which was reported by Hurt and Hauska (1981) and by Lemaire et al (1986). Many peroxidases are heme-containing enzymes that efficiently catalyse substrate oxidations using H 2 O 2 .…”

Section: Introductionmentioning

confidence: 90%

The peroxidase activity of cytochrome b6f complex from spinach chloroplasts

Chen¹,

Hou²,

et al. 2010

Photosynt.

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The cytochrome b 6 f (Cyt b 6 f) complex, which functions as a plastoquinol-plastocyanin oxidoreductase and mediates the linear electron flow between photosystem II (PSII) and photosystem I (PSI) and the cyclic electron flow around PSI, was isolated from spinach (Spinacia oleracea L.) chloroplasts using n-octyl-β-D-glucopyranoside (β-OG). The preparation was also able to catalyze the peroxidase-like reaction in the presence of hydrogen peroxide (H 2 O 2 ) and guaiacol. The optimal conditions for peroxidase activity of the preparation included: pH 3.6, ionic strength 0.1, and temperature 35ºC. The apparent Michaelis constant (K m ) values for H 2 O 2 and guaiacol were 50 mM and 2 mM, respectively. The bimolecular rate constant (k obs ) was about 26 M -1 s -1 and the turnover number (K cat ) was about 60 min -1 (20 mM guaiacol, 100 mM sodium phosphate, pH 3.6, 25°C, [H 2 O 2 ]<100mM). These parameters were similar to those of several other heme-containing proteins, such as myoglobin and Cyt c.Additional key words: cytochrome b 6 f complex; guaiacol; heme-containing protein; hydrogen peroxide; peroxidase activity.

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A Cytochrome f/b₆ Complex of Five Polypeptides with Plastoquinol‐Plastocyanin‐Oxidoreductase Activity from Spinach Chloroplasts

Cited by 396 publications

References 56 publications

Metastable radical state, nonreactive with oxygen, is inherent to catalysis by respiratory and photosynthetic cytochromes bc ₁ / b ₆ f

Metastable radical state, nonreactive with oxygen, is inherent to catalysis by respiratory and photosynthetic cytochromes bc ₁ / b ₆ f

Functional Characterization of the Small Regulatory Subunit PetP from the Cytochrome b₆f Complex in Thermosynechococcus elongatus

The peroxidase activity of cytochrome b<sub>6</sub>f complex from spinach chloroplasts

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A Cytochrome f/b6 Complex of Five Polypeptides with Plastoquinol‐Plastocyanin‐Oxidoreductase Activity from Spinach Chloroplasts

Cited by 396 publications

References 56 publications

Metastable radical state, nonreactive with oxygen, is inherent to catalysis by respiratory and photosynthetic cytochromes bc 1 / b 6 f

Metastable radical state, nonreactive with oxygen, is inherent to catalysis by respiratory and photosynthetic cytochromes bc 1 / b 6 f

Functional Characterization of the Small Regulatory Subunit PetP from the Cytochrome b6f Complex in Thermosynechococcus elongatus

The peroxidase activity of cytochrome b<sub>6</sub>f complex from spinach chloroplasts

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A Cytochrome f/b₆ Complex of Five Polypeptides with Plastoquinol‐Plastocyanin‐Oxidoreductase Activity from Spinach Chloroplasts

Metastable radical state, nonreactive with oxygen, is inherent to catalysis by respiratory and photosynthetic cytochromes bc ₁ / b ₆ f

Metastable radical state, nonreactive with oxygen, is inherent to catalysis by respiratory and photosynthetic cytochromes bc ₁ / b ₆ f

Functional Characterization of the Small Regulatory Subunit PetP from the Cytochrome b₆f Complex in Thermosynechococcus elongatus