Characterization of Four Covalently-Linked Yeast Cytochrome c/Cytochrome c Peroxidase Complexes:  Evidence for Electrostatic Interaction between Bound Cytochrome c Molecules

Nakani, Siddhartha; Vitello, Lidia B.; Erman, James E.

doi:10.1021/bi061662p

Cited by 9 publications

(23 citation statements)

References 22 publications

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“…However, in the extreme case of this rigid limit, if the site constants were equal to the measured thermodynamic affinity constants (K 1 ≈ K I and K 2 ≈ K II ), then repulsion would be zero, and this is ruled out by the measurements of Erman and coworkers, 42 as noted above. This requires that the actual site-binding constant K 2 must be larger than the measured value of K II = 10 4 , although still enough smaller than K 1 that the binary is predominantly in the tightly-binding form in accordance with the NMR studies.…”

Section: Discussionmentioning

confidence: 94%

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Control of Cyclic Photoinitiated Electron Transfer between Cytochrome c Peroxidase (W191F) and Cytochrome c by Formation of Dynamic Binary and Ternary Complexes

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Hoffman

2015

Biochemistry

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Extensive studies of the physiological protein—protein electron-transfer (ET) complex between yeast cytochrome c peroxidase (CcP) and cytochrome c (Cc) have left unresolved questions about how formation/dissociation of binary and ternary complexes influence ET. We probe this issue through study of the photocycle of ET between Zn-ProtoporphyrinIX-substituted CcP(W191F) (ZnPCcP) and Cc. Photoexcitation of ZnPCcP in complex with Fe3+Cc, initiates the photocycle: charge-separation ET [3ZnPCcP, Fe3+Cc]→[ZnP+CcP, Fe2+Cc] followed by charge recombination, [ZnP+CcP, Fe2+Cc] → [ZnPCcP, Fe3+Cc]. The W191F mutation eliminates fast hole hopping through W191, enhancing accumulation of charge-separated intermediate and extending the timescale for binding/dissociation of the charge-separated complex. Both triplet quenching and the charge-separated intermediate were monitored during titrations of ZnPCcP with Fe3+Cc, Fe2+Cc, and redox-inert CuCc. The results require a photocycle that includes dissociation/recombination of the charge-separated binary complex and a charge-separated ternary complex, [ZnP+CcP, Fe2+Cc, Fe3+Cc]. The expanded kinetic scheme formalizes earlier proposals of “substrate-assisted product dissociation” within the photocycle. The measurements yield the thermodynamic affinity constants for binding the first and second Cc: KI = 10−7 M−1, KII = 10−4 M−1. However, two-site analysis of the thermodynamics of formation of the ternary reveals that Cc binds at the weaker-binding site with much greater affinity than previously recognized, and places upper bounds on the contributions of repulsion between the two Cc of the ternary complex. In conjunction with recent NMR studies, the analysis further suggests a dynamic view of the ternary complex, wherein neither Cc necessarily faithfully adopts the crystal-structure configuration because of Cc-Cc repulsion.

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Section: Discussionmentioning

confidence: 94%

“…In fact, calculations and experiments have confirmed that the two Cc cannot bind independently, and that the stability of the ternary is diminished by electrostatic repulsions between the two positively charged Cc (anti-cooperative binding). 41, 42 …”

Section: Discussionmentioning

confidence: 99%

Control of Cyclic Photoinitiated Electron Transfer between Cytochrome c Peroxidase (W191F) and Cytochrome c by Formation of Dynamic Binary and Ternary Complexes

Page

Hoffman

2015

Biochemistry

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“…5a ). Previous experimental studies suggested that electrostatic repulsion between two Cc molecules in the ternary complex accounts for the drastic difference in the affinity constants for the first and second binding steps 37 38 39 . Difference in binding energies for the high- and low-affinity sites (ΔΔ G ) provides the upper limit for the electrostatic repulsion energy (Δ G Φ ), a presumed dominant term in the energy penalty for such ‘anticooperative' binding.…”

Section: Resultsmentioning

confidence: 99%

The low-affinity complex of cytochrome c and its peroxidase

2015

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The complex of yeast cytochrome c peroxidase and cytochrome c is a paradigm of the biological electron transfer (ET). Building on seven decades of research, two different models have been proposed to explain its functional redox activity. One postulates that the intermolecular ET occurs only in the dominant, high-affinity protein–protein orientation, while the other posits formation of an additional, low-affinity complex, which is much more active than the dominant one. Unlike the high-affinity interaction—extensively studied by X-ray crystallography and NMR spectroscopy—until now the binding of cytochrome c to the low-affinity site has not been observed directly, but inferred mainly from kinetics experiments. Here we report the structure of this elusive, weak protein complex and show that it consists of a dominant, inactive bound species and an ensemble of minor, ET-competent protein–protein orientations, which summarily account for the experimentally determined value of the ET rate constant.

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“…This hypothesis is supported by the cytochrome c/CcP cross-linking studies of Poulos and coworkers (28,45,46) as well as those of Nakani and colleagues (29,30). Pappa and Poulos (45) and Papa et al (46) used site-directed mutagenesis to engineer specific cysteine residues into both yeast cytochrome c and CcP to covalently attach cytochrome c to the Pelletier/Kraut binding site through a CcP Cys-290/cytochrome c Cys-73 disulfide bond.…”

Section: Relation To Other Studiesmentioning

confidence: 86%

Effect of Single-Site Charge-Reversal Mutations on the Catalytic Properties of Yeast Cytochrome c Peroxidase: Mutations near the High-Affinity Cytochrome c Binding Site

et al. 2007

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Fifteen single-site charge-reversal mutations of yeast cytochrome c peroxidase (CcP) have been constructed in order to determine the effect of localized charge on the catalytic properties of the enzyme. The mutations are located on the front face of CcP, near the cytochrome c binding site identified in the crystallographic structure of the yeast cytochrome c/CcP complex (Pelletier and Kraut (1992) Science 258, 1748-1755. The mutants are characterized by absorption spectroscopy and hydrogen peroxide reactivity at both pH 6.0 and 7.5 and by steady-state kinetic studies using recombinant yeast iso-1 ferrocytochrome c(C102T) as substrate at pH 7.5. Some of the chargereversal mutations cause detectable changes in the absorption spectrum, especially at pH 7.5, reflecting changes in the equilibrium between penta-and hexa-coordinate heme species in the enzyme. An increase in the amount of hexa-coordinate heme in the mutant enzymes correlates with an increase in the fraction of enzyme that does not react with hydrogen peroxide. Steady-state velocity measurements indicate that five of the fifteen mutations cause large increases in the Michaelis constant (R31E, D34K, D37K, E118K, and E290K). These data support the hypothesis that the cytochrome c/CcP complex observed in the crystal is the dominant catalytically active complex in solution.Cytochrome c peroxidase, CcP 1 , is a detoxification enzyme localized between the inner and outer membranes of yeast mitochondria (1). CcP decreases toxic levels of hydrogen peroxide by catalyzing its reduction to water using ferrocytochrome c (2). The catalytic mechanism involves oxidation of the native enzyme by hydrogen peroxide to an enzyme intermediate called CcP Compound I, CcP-I. CcP-I contains two oxidized sites, an oxyferryl Fe(IV) heme group and a tryptophan π-cation radical located within van der Waals distance of the heme. Interaction with, and electron transfer from, ferrocytochrome c reduces CcP-I back to the native state via a second enzyme intermediate, CcP Compound II, CcP-II, completing the catalytic cycle. Since 1980, when the three-dimensional structure of CcP was first reported (3,4), CcP has played an important role in elucidating the structural basis for heme protein reactivity, † This work was supported in part by a grant from the National Institutes of Health (R15 GM59740). * Corresponding author. Phone: (815) . E-mail: jerman@niu.edu. 1 Abbreviations: Mutations in the amino acid sequences of either CcP or cytochrome c are indicated by using the one letter code for the amino acid residue in the wild-type protein, followed by the residue number and the one letter code for the amino acid residue in the mutant protein, i.e., C102T represents a mutant in which a threonine residue replaces the cysteine residue at position 102 of the wildtype protein; CcP, generic abbreviation for cytochrome c peroxidase whatever the source; yCcP, authentic yeast cytochrome c peroxidase isolated from bakers' yeast, Saccharomyces cervisiae; rCcP, recombinant CcP expressed in E. ...

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Characterization of Four Covalently-Linked Yeast Cytochrome c/Cytochrome c Peroxidase Complexes: Evidence for Electrostatic Interaction between Bound Cytochrome c Molecules

Abstract: Four covalent complexes between recombinant yeast cytochrome c and cytochrome c peroxidase (rCcP) were synthesized via disulfide bond formation using specifically designed protein mutants [Papa, H. S., and Poulos, T.

Cited by 9 publications

References 22 publications

Control of Cyclic Photoinitiated Electron Transfer between Cytochrome c Peroxidase (W191F) and Cytochrome c by Formation of Dynamic Binary and Ternary Complexes

Control of Cyclic Photoinitiated Electron Transfer between Cytochrome c Peroxidase (W191F) and Cytochrome c by Formation of Dynamic Binary and Ternary Complexes

The low-affinity complex of cytochrome c and its peroxidase

Effect of Single-Site Charge-Reversal Mutations on the Catalytic Properties of Yeast Cytochrome c Peroxidase: Mutations near the High-Affinity Cytochrome c Binding Site

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