Design of a Ruthenium-Cytochrome c Derivative to Measure Electron Transfer to the Initial Acceptor in Cytochrome c Oxidase

Geren, Lois; Beasley, James R.; Fine, Bryan R.; Saunders, Aleister J.; Hibdon, Sharon; Pielak, Gary J.; Durham, Bill; Millett, Francis

doi:10.1074/jbc.270.6.2466

Cited by 93 publications

(172 citation statements)

References 65 publications

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“…The identity of the additional heme species as heme O was unequivocally confirmed by heme extraction and subsequent HPLC analysis using heme extracts from wild-type P. denitrificans cytochrome aa 3 and E. coli cytochrome bo 3 as controls (see Fig. 3B).…”

Section: Resultsmentioning

confidence: 98%

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Mutation of Arg-54 Strongly Influences Heme Composition and Rate and Directionality of Electron Transfer in Paracoccus denitrificans Cytochrome c Oxidase

Kannt¹,

Pfitzner²,

Ruitenberg³

et al. 1999

Journal of Biological Chemistry

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The effect of a single site mutation of Arg-54 to methionine in Paracoccus denitrificans cytochrome c oxidase was studied using a combination of optical spectroscopy, electrochemical and rapid kinetics techniques, and time-resolved measurements of electrical membrane potential. The mutation resulted in a blue-shift of the heme a ␣-band by 15 nm and partial occupation of the low-spin heme site by heme O. Additionally, there was a marked decrease in the midpoint potential of the low-spin heme, resulting in slow reduction of this heme species. A stopped-flow investigation of the reaction with ferrocytochrome c yielded a kinetic difference spectrum resembling that of heme a 3 . This observation, and the absence of transient absorbance changes at the corresponding wavelength of the low-spin heme, suggests that, in the mutant enzyme, electron transfer from Cu A to the binuclear center may not occur via heme a but that instead direct electron transfer to the high-spin heme is the dominating process. This was supported by charge translocation measurements where ⌬ generation was completely inhibited in the presence of KCN. Our results thus provide an example for how the interplay between protein and cofactors can modulate the functional properties of the enzyme complex.

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Section: Resultsmentioning

confidence: 98%

“…Cytochrome c-Cu A electron transfer occurs at a rate of 70,000 s Ϫ1 (2,3). The rate-limiting step for reduction of Cu A , however, is the binding of cytochrome c that is strongly dependent on ionic strength (4,5).…”

mentioning

confidence: 99%

Mutation of Arg-54 Strongly Influences Heme Composition and Rate and Directionality of Electron Transfer in Paracoccus denitrificans Cytochrome c Oxidase

Kannt¹,

Pfitzner²,

Ruitenberg³

et al. 1999

Journal of Biological Chemistry

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“…4C shows the oxidation kinetics of cytochrome c at the highest [O 2 ] studied here. The quality of the fit was clearly improved when using two exponentials rather than one, pointing to the occurrence of distinct, kinetically limiting, steps for the fast and slow components as the diffusion-limited oxidation of cytochrome c has been shown to be a pseudofirst order process (43,48). This multiphasic behavior thus discloses at least two different types of cytochrome c, the fastest fraction being oxidized with a rate constant of ∼3.6 10 3 s −1 (time constant ∼280 μs).…”

Section: Discussionmentioning

confidence: 99%

“…Despite the kinetic limitation resulting from the limited [O 2 ] which could be used, the possibility to characterize the kinetics of cytochrome c oxidation at 551 nm provides a powerful means to assess the degree of binding to its membrane-bound electron acceptor. Indeed, electron transfer from reduced, bound cytochrome c to oxidized Cu A of CcOx has been shown to occur in vitro with rates ranging from 7 10 3 to 6 10 4 s −1 (42,43). As another example, in bacterial caa 3 -type oxidases which contain a c-type cytochrome subunit, the oxidation of heme c following photoactivation develops in the 30 to 240 μs range (44)(45)(46).…”

Section: Light-activation Of the Respiratory Chain In The Presence Ofmentioning

confidence: 99%

Questioning the functional relevance of mitochondrial supercomplexes by time-resolved analysis of the respiratory chain

Trouillard

Meunier

Rappaport

2011

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

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Mitochondria are the powerhouses of eukaryotic cells as they feed metabolism with its major substrate. Oxidative-phosphorylation relies on the generation, by an electron/proton transfer chain, of an electrochemical transmembrane potential utilized to synthesize ATP. Although these fundamental principles are not a matter of debate, the emerging picture of the respiratory chain diverges from the linear and fluid scheme. Indeed, a growing number of pieces of evidence point to membrane compartments that possibly restrict the diffusion of electron carriers, and to supramolecular assembly of various complexes within various kinds of supercomplexes that modulate the thermodynamic and kinetic properties of the components of the chain. Here, we describe a method that allows the unprecedented time-resolved study of the respiratory chain in intact cells that is aimed at assessing these hypotheses. We show that, in yeast, cytochrome c is not trapped within supercomplexes and encounters no particular restriction to its diffusion which questions the functional relevance of these supramolecular edifices.bioenergetics | electon transfer | respiration | compartmentalization

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“…[1][2][3] Due to their wide availability, robust structure, and ease of handling, these proteins have served as model systems to study the interplay of protein structure, dynamics, and function. [4][5][6][7][8][9][10][11][12][13] Cytochrome c 552 from Hydrogenobacter thermophilus (Ht-cyt c 552 ) [14][15][16][17] was recently shown to be a structurally unique example within the cyt c 8 family of class I 18 cyt c's. Proteins in the cyt c 8 structural family, for example, Pseudomonas cyt c 551 's, [19][20][21] typically have an asparagine residue at position 64 (Asn64) that is situated to donate a hydrogen bond to a methionine at position 61 (Met61), which occupies the sixth coordination site to the heme ( Figure 1a; sequence numbering is based on the Pseudomonas aeruginosa cyt c 551 sequence).…”

Section: Introductionmentioning

confidence: 99%

Cytochrome c₅₅₂Mutants: Structure and Dynamics at the Active Site Probed by Multidimensional NMR and Vibration Echo Spectroscopy

et al. 2006

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Spectrally resolved infrared stimulated vibrational echo experiments are used to measure the vibrational dephasing of a CO ligand bound to the heme cofactor in two mutated forms of the cytochrome c 552 from Hydrogenobacter thermophilus. The first mutant (Ht-M61A) is characterized by a single mutation of Met61 to an Ala (Ht-M61A), while the second variant is doubly modified to have Gln64 replaced by an Asn in addition to the M61A mutation (Ht-M61A/Q64N). Multidimensional NMR experiments determined that the geometry of residue 64 in the two mutants is consistent with a non-hydrogen-bonding and hydrogen-bonding interaction with the CO ligand for Ht-M61A and Ht-M61A/Q64N, respectively. The vibrational echo experiments reveal that the shortest time scale vibrational dephasing of the CO is faster in the Ht-M61A/ Q64N mutant than that in Ht-M61A. Longer time scale dynamics, measured as spectral diffusion, are unchanged by the Q64N modification. Frequency-frequency correlation functions (FFCFs) of the CO are extracted from the vibrational echo data to confirm that the dynamical difference induced by the Q64N mutation is primarily an increase in the fast (hundreds of femtoseconds) frequency fluctuations, while the slower (tens of picoseconds) dynamics are nearly unaffected. We conclude that the faster dynamics in Ht-M61A/Q64N are due to the location of Asn64, which is a hydrogen bond donor, above the heme-bound CO. A similar difference in CO ligand dynamics has been observed in the comparison of the CO derivative of myoglobin (MbCO) and its H64V variant, which is caused by the difference in axial residue interactions with the CO ligand. The results suggest a general trend for rapid ligand vibrational dynamics in the presence of a hydrogen bond donor.

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Design of a Ruthenium-Cytochrome c Derivative to Measure Electron Transfer to the Initial Acceptor in Cytochrome c Oxidase

Cited by 93 publications

References 65 publications

Mutation of Arg-54 Strongly Influences Heme Composition and Rate and Directionality of Electron Transfer in Paracoccus denitrificans Cytochrome c Oxidase

Mutation of Arg-54 Strongly Influences Heme Composition and Rate and Directionality of Electron Transfer in Paracoccus denitrificans Cytochrome c Oxidase

Questioning the functional relevance of mitochondrial supercomplexes by time-resolved analysis of the respiratory chain

Cytochrome c₅₅₂Mutants: Structure and Dynamics at the Active Site Probed by Multidimensional NMR and Vibration Echo Spectroscopy

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Design of a Ruthenium-Cytochrome c Derivative to Measure Electron Transfer to the Initial Acceptor in Cytochrome c Oxidase

Cited by 93 publications

References 65 publications

Mutation of Arg-54 Strongly Influences Heme Composition and Rate and Directionality of Electron Transfer in Paracoccus denitrificans Cytochrome c Oxidase

Mutation of Arg-54 Strongly Influences Heme Composition and Rate and Directionality of Electron Transfer in Paracoccus denitrificans Cytochrome c Oxidase

Questioning the functional relevance of mitochondrial supercomplexes by time-resolved analysis of the respiratory chain

Cytochrome c552Mutants: Structure and Dynamics at the Active Site Probed by Multidimensional NMR and Vibration Echo Spectroscopy

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Product

Resources

About

Cytochrome c₅₅₂Mutants: Structure and Dynamics at the Active Site Probed by Multidimensional NMR and Vibration Echo Spectroscopy