ESEEM and ENDOR studies of the Rieske iron-sulphur protein in bovine heart mitochondrial membranes (original) (raw)
Related papers
Biochemistry, 1991
Electron spin echo envelope modulation (ESEEM) experiments performed on the Rieske Fe-S clusters of the cytochrome b$complex of spinach chloroplasts and of the cytochrome bcl complexes of Rhodospirillum rubrum, Rhodobacter sphaeroides R-26, and bovine heart mitochondria show modulation components resulting from two distinct classes of I4N ligands. At the g = 1.92 region of the Rieske EPR spectrum of the cytochrome b$complex, the measured hyperfine couplings for the two classes of coupled nitrogens are A I = 4.6 M H z and A2 = 3.8 MHz. Similar couplings are observed for the Rieske centers in thc three cytochrome bc, 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 Biochemistry 28, 4861-48711. The Rieske Fe-S cluster lacks modulation components from a weakly coupled peptide nitrogen observed in water-soluble spinach ferredoxin. Trcatment 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.
Proceedings of the National Academy of Sciences, 1998
We have analyzed crystal structures of cytochrome bc 1 complexes with electron transfer inhibitors bound to the ubiquinone binding pockets Q i and/or Q o in the cytochrome b subunit. The presence or absence of the Q i inhibitor antimycin A did not affect the binding of the Q o inhibitors. Different subtypes of Q o inhibitors had dramatically different effects on the mobility of the extramembrane domain of the iron–sulfur protein (ISP): Binding of 5-undecyl-6-hydroxy-4,7-dioxobenzothiazol and stigmatellin (subtype Q o –II and Q o –III, respectively) led to a fixation of the ISP domain on the surface of cytochrome b , whereas binding of myxothiazol and methoxyacrylate-stilbene (subtype Q o –I) favored release of this domain. The native structure has an empty Q o pocket and is intermediate between these extremes. On the basis of these observations we propose a model of quinone oxidation in the bc 1 complex, which incorporates fixed and loose states of the ISP as features important for ...
Journal of Biological Chemistry, 2002
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 for 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 ␦ 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 ␦ 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.
Biochemistry, 2007
The catalytic sites of beef heart mitochondrial F1-ATPase were studied by electron spin echo envelope modulation (ESEEM) spectroscopy, using Mn(II) as a paramagnetic probe, which replaces the naturally occurring Mg(II), maintaining the enzyme catalytic activity. F1-ATPase was purified from beef heart mitochondria. A protein still containing three endogenous nucleotides, named MF1(1,2), is obtained under milder conditions, whereas a harsher treatment gives a fully depleted F1, named MF1(0,0). Several samples were prepared, loading MF1(0,0) or MF1(1,2) with Mn(II) or Mn II ADP in both substoichiometric and excess amounts. When MF1(1,2) is loaded with Mn(II) in a 1:0.8 ratio, the FT-ESEEM spectrum shows evidence of a nitrogen interacting with the metal, while this interaction is not present in MF1(0,0) + Mn(II) in a 1:0.8 ratio. However, when MF1(0,0) is loaded with 2.4 Mn(II), the FT-ESEEM spectrum shows a metal-nitrogen interaction resembling that present in MF1(1,2) + Mn(II) in a 1:0.8 ratio. These results strongly support the role of the metal alone in shaping and structuring the catalytic sites of the enzyme. When substoichiometric ADP is added to MF1(1,2) preloaded with 0.8 equiv of Mn(II), the ESEEM spectra show evidence of a phosphorus nucleus coupled to the metal, indicating that the nucleotide phosphate binding to Mn(II) occurs in a catalytic site. Generally, 14 N coordination to the metal is clearly identified in the ESEEM spectra of all the samples containing more than one metal equivalent. One point of note is that the relevant nitrogen-containing ligand(s), responsible for the signals in the ESEEM spectra, has not yet been identified in the available X-ray structures. † This work was supported by the University of Padova through the project "Studio con risonanze di Spin elettronico dell'enzima F1" (Cdr. A.0EE00.96) and by Regione Friuli Venezia Giulia (legge regionale 11/2003).
Biochimica et Biophysica Acta (BBA) - Bioenergetics, 1978
The iron-sulfur protein present in the mitochondrial outer membrane has been partially purified from beef kidney cortex mitochondria be means of selective solubilization followed by DEAE-cellulose chromatography. The EPR spectrum of the iron-sulfur protein with g-values at 2.01, 1.94 and 1.89 was well resolved up to 200 K which is unusual for an iron-sulfur protein. Analyses confirmed a center with two iron and two labile sulfur atoms in the protein. By measuring the effect of oxidation-reduction potential on the EPR signal amplitude, midpoint potentials at pH 7.2 were determined both for the purified ironsulfur protein, +75 (+5) mV, and in prepared mitochondrial outer membrane, +62 (+6) mV. At pH 8.2 slightly lower values were indicated, +62 and 52 mV, respectively. The oxidation-reduction equilibrium involved a one electron transfer. A functional relationship to the rotenone-insensitive NADH-cytochrome c oxidoreductase in the mitochondrial outer membrane is suggested. Both this activity and the iron-sulfur center were sensitive to acidities slightly below pH 7 in contrast to the iron-sulfur centers of the inner membrane.
Cytochrome c interaction with the mitochondrial membrane: A spin label study
Biochimica et Biophysica Acta (BBA) - Bioenergetics, 1972
Horse-heart ferrocytochrome c has been labeled with N-(2,2,5,5-tetramethyl-3pyrrolidinyl-I-oxyl) iodoacetamide at methionine-65. The paramagnetic resonance spectrum of labeled ferricytochrome c indicates a weak immobilization of the radical (ze = 9.3" lO-I° sec) which becomes stronger upon binding of labeled cytochrome c to cytochrome c-depleted mitochondrial membranes (Ze-3.3"1o 9 sec). The hyperfine coupling constant remains, however, unchanged (16. 7 ± o.I gauss) indicating that the cytochrome c binding site is highly polar. The region where cytochrome c is bound to the membrane is insensitive to large variations of medium viscosity.
Biochimica et biophysica acta, 1975
The composite ESR spectra of nitroxide-maleimide spin-labelled albumin and mitochondrial membranes were investigated. It was found that the mobile spectrum (so called "weakly immobilized") is due to the probe free in solution; it could be removed by dialysis, by lowering the ratio of spin label to albumin or by using acid pH values. Similar degrees of mobility were noted with spectra of spin label in solutions with viscosities similar to that of albumin. The immobile spectrum (so called "strongly immobilized") is due to the binding of N-(1-oxyl-2,2,6,6-tetramethyl-4-piperidinyl)-maleimide to the proteins. Studies with modified albumins have shown that in this binding the free SH and amino groups are involved, but the three-dimensional structure offering conditions for hydrophobic binding is also required. The spectral parameters useful for evaluating conformational changes of proteins and an application to mitochondrial membranes are described.
European Journal of Biochemistry, 1992
Protein/phospholipid interactions in the solubilized mitochondrial ubihydroquinone : cytochromec oxidoreductase (bcl complex) were studied by spin-label electron-spin resonance and by " P-NMR spectroscopy. Spin-labelled phospholipids were employed to probe the relative binding afinities of a number of phospholipids with regard to the significance of phospholipids for the activity and stability of this multisubunit complex. The protein was titrated with spin-labelled cardiolipin (1,3bisphosphatidyl-sn-glycerol) and with the spin-labelled analogues of PtdCho and PtdEtn, both of which have been shown recently to elicit a substantial increase in electron-transport activity [Schagger, 1301. A simplified distribution model showed that neutral phospholipids have much lowcr protein affinity than cardiolipin. In contrast to the transient weak lipid binding detected by spin-label electronspin resonance, 31P NMR revealed a tightly bound cardiolipin portion, even after careful delipidation of the complex, Considerable line narrowing was observed after phospholipase A, digestion of the bound cardiolipin, whereas addition of SDS resulted in complete release. Relative proportions and line widths of mobile and immobilized lipids were obtained by deconvoluting the partially overlapping signals. The current results are discussed with reference to similar findings with other mitochondrial membrane proteins. It is assumed that activation by neutral phospholipids reflects a generalized effect on the protein conformation. Cardiolipin binding is believed to be important for the structural integrity of the mitochondrial protein complexes.
Membrane potential and surface potential in mitochondria. Binding of a cationic spin probe
Biochimica et Biophysica Acta (BBA) - Bioenergetics, 1984
The effects of surface potential and transmembrane potential on the binding and fluorescence of 1-anilinonaphthalene-8-sulfonate (ANS) in suspensions of rat liver mitochondria was investigated. The binding of ANS is characterized by two classes of binding site: a high affinity (Kd = 10-50 @M), low capacity (n = 3-8 nmol/mg of protein) class in which bound ANS fluoresces strongly, and a low affinity (>500 pM), high capacity (>50 nmol/mg of protein) class with little fluorescence. The dissociation constant, Kd, of the high affinity site strongly depends on the surface potential of the external surface of the inner mitochondrial membrane. Hence, the binding and fluorescence of ANS can be used to estimate the surface potential. The dependence of ANS binding on the medium salt concentration is compatible with the Gouy-Chapman theory and allows accurate determination of surface potential and surface charge. The generation of transmembrane potential, either by oxidizable substrates, ATP, or POtassium gradient leads to a decrease in the fluorescence. This decrease is the result of reduced ANS binding. However, the external surface potential as estimated from the charge screening effect of salt solutions is unchanged in energized membranes. The extent of decreased fluorescence correlates reasonably well with the magnitude of the transmembrane potential. The potential-induced quenching depends on pre-equilibration of ANS with the mitochondria, suggesting that the response is due to extrusion of ANS from the mitochondrial matrix. These findings do not support the suggestion that ANS quenching in energized mitochondria is due to an increase in the negative surface charge of the cytosolic surface of the inner membrane. The results are compatible with the suggestion that the response to energization is largely due to the formation of A$. However, because of the complex nature of the ANS response, it is concluded that neither the magnitude of surface potential nor the magnitude of membrane potential can be determined from the ANS response in energized mitochondria. Substrate-induced "energization" of mitochondria induces the quenching of fluorescence of the anionic fluorescent probe of Pathology, Hanemann University, Broad and Vine Sts., Philadelphia, PA 19102. ANSI (1). This discovery, over 13 years ago, was followed by a rapid spread of the use of ANS as a probe for energization in mitochondria and in many other membrane systems (for review, see Ref.
FEBS Letters, 1994
Treatment of bovine heart ubiquinol-cytochrome c oxidoreductase (complex III, bc, complex) with ethoxyformic anhydride (EFA) inhibits electron transfer between cytochromes b and c, wagi et al., Biochemistry 21(1982) 4777-47823. This paper shows that EFA alters the EPR lineshape of the Rieske iron-sulfur cluster in complex III and in the isolated Rieske protein without a significant decrease of spin concentration. The effect of EFA on the Rieske iron-sulfur cluster is competitive with that of Q, site inhibitors, such as stigmatellin, and is completely reversed by hydroxylamine. These results are consistent with the possible ethoxyformylation by EFA of histidine ligands of the Rieske iron-sulfur cluster at the non-iron binding imidazole nitrogens.