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Title: Plasmon waveguide resonance spectroscopic evidence fordifferential binding of oxidized and reduced rhodobacter capsulatuscytochrome c(2) to the cytochrome bc(1) complex mediated by theconformation of the rieske iron-sulfur protein

Abstract

The dissociation constants for the binding of Rhodobactercapsulatus cytochrome c2 and its K93P mutant to the cytochrome bc1complex embedded in a phospholipid bilayer were measured by plasmonwaveguide resonance spectroscopy in the presence and absence of theinhibitor stigmatellin. The reduced form of cytochrome c2 strongly bindsto reduced cytochrome bc1 (Kd = 0.02 M) but binds much more weakly to theoxidized form (Kd = 3.1 M). In contrast, oxidized cytochrome c2 binds tooxidized cytochrome bc1 in a biphasic fashion with Kd values of 0.11 and0.58 M. Such a biphasic interaction is consistent with binding to twoseparate sites or conformations of oxidized cytochrome c2 and/orcytochrome bc1. However, in the presence of stigmatellin, we find thatoxidized cytochrome c2 binds to oxidized cytochrome bc1 in a monophasicfashion with high affinity (Kd = 0.06 M) and reduced cytochrome c2 bindsless strongly (Kd = 0.11 M) but ~;30-fold more tightly than in theabsence of stigmatellin. Structural studies with cytochrome bc1, with andwithout the inhibitor stigmatellin, have led to the proposal that theRieske protein is mobile, moving between the cytochrome b and cytochromec1 components during turnover. In one conformation, the Rieske proteinbinds near the heme of cytochrome c1, while the cytochrome c2 bindingsite is also near the cytochromemore » c1 heme but on the opposite side fromthe Rieske site, where cytochrome c2 cannot directly interact withRieske. However, the inhibitor, stigmatellin, freezes the Rieske proteiniron-sulfur cluster in a conformation proximal to cytochrome b and distalto cytochrome c1. We conclude from this that the dual conformation of theRieske protein is primarily responsible for biphasic binding of oxidizedcytochrome c2 to cytochrome c1. This optimizes turnover by maximizingbinding of the substrate, oxidized cytochrome c2, when the iron-sulfurcluster is proximal to cytochrome b and minimizing binding of theproduct, reduced cytochrome c2, when it is proximal to cytochromec1.« less

Authors:
; ; ; ; ; ;
Publication Date:
Research Org.:
COLLABORATION - University ofArizona
OSTI Identifier:
929360
Report Number(s):
LBNL-62862
R&D Project: 864U1F; TRN: US0803724
DOE Contract Number:
DE-AC02-05CH11231
Resource Type:
Journal Article
Resource Relation:
Journal Name: Biochemistry; Journal Volume: 46; Journal Issue: 24; Related Information: Journal Publication Date: Jun 19 2007
Country of Publication:
United States
Language:
English
Subject:
59; AFFINITY; CYTOCHROMES; DISSOCIATION; HEME; MUTANTS; PHOSPHOLIPIDS; PLASMONS; PROTEINS; RESONANCE; SPECTROSCOPY; WAVEGUIDES; plasmon waveguide resonance; photosynthetic reaction center; cytochrome bc1 complex; cytochrome c2; Rieske iron-sulfur protein; quinone; cytochrome b.

Citation Formats

Devanathan, S., Salamon, Z., Tollin, G., Fitch, J.C., Meyer,T.E., Berry, E.A., and Cusanovich, M.A. Plasmon waveguide resonance spectroscopic evidence fordifferential binding of oxidized and reduced rhodobacter capsulatuscytochrome c(2) to the cytochrome bc(1) complex mediated by theconformation of the rieske iron-sulfur protein. United States: N. p., 2007. Web. doi:10.1021/bi602649u.
Devanathan, S., Salamon, Z., Tollin, G., Fitch, J.C., Meyer,T.E., Berry, E.A., & Cusanovich, M.A. Plasmon waveguide resonance spectroscopic evidence fordifferential binding of oxidized and reduced rhodobacter capsulatuscytochrome c(2) to the cytochrome bc(1) complex mediated by theconformation of the rieske iron-sulfur protein. United States. doi:10.1021/bi602649u.
Devanathan, S., Salamon, Z., Tollin, G., Fitch, J.C., Meyer,T.E., Berry, E.A., and Cusanovich, M.A. Tue . "Plasmon waveguide resonance spectroscopic evidence fordifferential binding of oxidized and reduced rhodobacter capsulatuscytochrome c(2) to the cytochrome bc(1) complex mediated by theconformation of the rieske iron-sulfur protein". United States. doi:10.1021/bi602649u.
@article{osti_929360,
title = {Plasmon waveguide resonance spectroscopic evidence fordifferential binding of oxidized and reduced rhodobacter capsulatuscytochrome c(2) to the cytochrome bc(1) complex mediated by theconformation of the rieske iron-sulfur protein},
author = {Devanathan, S. and Salamon, Z. and Tollin, G. and Fitch, J.C. and Meyer,T.E. and Berry, E.A. and Cusanovich, M.A.},
abstractNote = {The dissociation constants for the binding of Rhodobactercapsulatus cytochrome c2 and its K93P mutant to the cytochrome bc1complex embedded in a phospholipid bilayer were measured by plasmonwaveguide resonance spectroscopy in the presence and absence of theinhibitor stigmatellin. The reduced form of cytochrome c2 strongly bindsto reduced cytochrome bc1 (Kd = 0.02 M) but binds much more weakly to theoxidized form (Kd = 3.1 M). In contrast, oxidized cytochrome c2 binds tooxidized cytochrome bc1 in a biphasic fashion with Kd values of 0.11 and0.58 M. Such a biphasic interaction is consistent with binding to twoseparate sites or conformations of oxidized cytochrome c2 and/orcytochrome bc1. However, in the presence of stigmatellin, we find thatoxidized cytochrome c2 binds to oxidized cytochrome bc1 in a monophasicfashion with high affinity (Kd = 0.06 M) and reduced cytochrome c2 bindsless strongly (Kd = 0.11 M) but ~;30-fold more tightly than in theabsence of stigmatellin. Structural studies with cytochrome bc1, with andwithout the inhibitor stigmatellin, have led to the proposal that theRieske protein is mobile, moving between the cytochrome b and cytochromec1 components during turnover. In one conformation, the Rieske proteinbinds near the heme of cytochrome c1, while the cytochrome c2 bindingsite is also near the cytochrome c1 heme but on the opposite side fromthe Rieske site, where cytochrome c2 cannot directly interact withRieske. However, the inhibitor, stigmatellin, freezes the Rieske proteiniron-sulfur cluster in a conformation proximal to cytochrome b and distalto cytochrome c1. We conclude from this that the dual conformation of theRieske protein is primarily responsible for biphasic binding of oxidizedcytochrome c2 to cytochrome c1. This optimizes turnover by maximizingbinding of the substrate, oxidized cytochrome c2, when the iron-sulfurcluster is proximal to cytochrome b and minimizing binding of theproduct, reduced cytochrome c2, when it is proximal to cytochromec1.},
doi = {10.1021/bi602649u},
journal = {Biochemistry},
number = 24,
volume = 46,
place = {United States},
year = {Tue May 22 00:00:00 EDT 2007},
month = {Tue May 22 00:00:00 EDT 2007}
}
  • In this report, the authors used in situ hybridization and somatic cell hybrid mapping to localize the human gene for the Rieske iron-sulfur protein. The assignment of the gene for the Rieske iron-sulfur protein to human chromosomes 22q13 and 19q12-q13.1 confirms that it is encoded by the nuclear genome. The localization of four subunits of complex III to different human chromosomes - 8, 16, and 22/19 - precludes the existence of a gene cluster for this complex within the human genome. 9 refs., 2 figs., 1 tab.
  • Electron spin echo envelope modulation (ESEEM) experiments performed on the Rieske Fe-S clusters of the cytochrome b{sub 6}f complex of spinach chloroplasts and of the cytochrome bc{sub 1} complexes of Rhodospirillum rubrum, Rhodobacter sphaeroides R-26, and bovine heart mitochondria show modulation components resulting from two distinct classes of {sup 14}N ligands. At the g = 1.92 region of the Rieske EPR spectrum of the cytochrome b{sub 6}f complex, the measured hyperfine couplings for the two classes of coupled nitrogens are A{sub 1} = 4.6 MHz and A{sub 2} = 3.8 MHz. Similar couplings are observed for the Rieske centers inmore » the three cytochrome bc{sub 1} complexes. These ESEEM results indicate a nitrogen coordination environment for these Rieske Fe-S centers that is similar to that of the Fe-S cluster of a bacterial dioxygenase enzyme with two coordinated histidine ligands. The Rieske Fe-S cluster lacks modulation components from a weakly coupled peptide nitrogen observed in water-soluble spinach ferredoxin. Treatment with the quinone analogue inhibitor DBMIB causes a shift in the Rieske EPR spectrum to g = 1.95 with no alteration in the magnetic couplings to the two nitrogen atoms. However, the ESEEM pattern of the DBMIB-altered Rieske EPR signal shows evidence of an additional weakly coupled nitrogen similar to that observed in the spinach ferrodoxin ESEEM patterns.« less
  • The bifurcated reaction at the Q(o)-site of the bc(1) complex provides the mechanistic basis of the proton pumping activity through which the complex conserves redox energy in the proton gradient. Structural information about the binding of quinone at the site is lacking, because the site is vacant in crystals of the native complexes. We now report the first structural characterization of the interaction of the native quinone occupant with the Rieske iron-sulfur protein in the bc(1) complex of Rhodobacter sphaeroides, using high resolution EPR. We have compared the binding configuration in the presence of quinone with the known structures formore » the complex with stigmatellin and myxothiazol. We have shown by using EPR and orientation-selective electron spin echo envelope modulation (ESEEM) measurements of the iron-sulfur protein that when quinone is present in the site, the isotropic hyperfine constant of one of the N(delta) atoms of a liganding histidine of the[2Fe-2S] cluster is similar to that observed when stigmatellin is present and different from the configuration in the presence of myxothiazol. The spectra also show complementary differences in nitrogen quadrupole splittings in some orientations. We suggest that the EPR characteristics, the ESEEM spectra, and the hyperfine couplings reflect a similar interaction between the iron-sulfur protein and the quinone or stigmatellin and that the N(delta) involved is that of a histidine (equivalent to His-161 in the chicken mitochondrial complex) that forms both a ligand to the cluster and a hydrogen bond with a carbonyl oxygen atom of the Q(o)-site occupant« less
  • Cytochrome c/sub 2/ and the detergent-solubilized cytochrome bc/sub 1/ complex, both from Rhodospirillum rubrum, form a tight complex at a low ionic strength that can be isolated by gel permeation chromatography. The dissociation constant of the complex is estimated to be 10/sup -6/ M or less. (/sup 14/C)-labelled and (/sup 3/H)-labelled acetic anhydride were used in the acetylation experiments, and the /sup 3/H//sup 14/C ratio was analyzed. The binding site for the cytochrome bc/sub 1/ complex on cytochrome c/sub 2/ was analyzed by differential acetylation of lysine residues in free and cytochrome bc/sub 1/ complex bound cytochrome c/sub 2/. Inmore » bound cytochrome c/sub 2/, three lysine residues at sequence positions 12, 13, and 97 were less reactive toward acetic anhydride. Lys/sup 13/, which is located above the exposed heme edge, was the least reactive, i.e., the most shielded by the cytochrome bc/sub 1/ complex. Correlating this information with the crystal structure of cytochrome c/sub 2/ indicates that the binding site for the cytochrome bc/sub 1/ complex on cytochrome c/sub 2/ involves a surface area above, and probably including, the exposed heme edge. This mode of binding is similar to that observed for horse cytochrome c interacting with the mitochrondrial cytochrome bc/sub 1/ complex. A simplified version of the method of differential chemical modification is presented.« less