The Interaction of the Rieske Iron-Sulfur Protein with Occupants of the Qo-site of the bc1 Complex, Probed by Electron Spin Echo Envelope Modulation (original) (raw)
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Biochemistry, 1999
Crystallographic structures for the mitochondrial ubihydroquinone:cytochrome c oxidoreductase (bc 1 complex) from different sources, and with different inhibitors in cocrystals, have revealed that the extrinsic domain of the iron sulfur subunit is not fixed [Zhang677-684], but moves between reaction domains on cytochrome c 1 and cytochrome b subunits. We have suggested that the movement is necessary for quinol oxidation at the Q o site of the complex. In this paper, we show that the electron-transfer reactions of the high-potential chain of the complex, including oxidation of the iron sulfur protein by cytochrome c 1 and the reactions by which oxidizing equivalents become available at the Q o site, are rapid compared to the rate-determining step. Activation energies of partial reactions that contribute to movement of the iron sulfur protein have been measured and shown to be lower than the high activation barrier associated with quinol oxidation. We conclude that the movement is not the source of the activation barrier. We estimate the occupancies of different positions for the iron sulfur protein from the crystallographic electron densities and discuss the parameters determining the binding of the iron sulfur protein in different configurations. The low activation barrier is consistent with a movement between these locations through a constrained diffusion. Apart from ligation in enzymesubstrate or inhibitor complexes, the binding forces in the native structure are likely to be eRT, suggesting that the mobile head can explore the reaction interfaces through stochastic processes within the time scale indicated by kinetic measurements.
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.
Biochimica et Biophysica Acta (BBA) - Bioenergetics, 2002
Since available structures of native bc 1 complexes show a vacant Q o -site, occupancy by substrate and product must be investigated by kinetic and spectroscopic approaches. In this brief review, we discuss recent advances using these approaches that throw new light on the mechanism. The rate-limiting reaction is the first electron transfer after formation of the enzyme -substrate complex at the Q o -site. This is formed by binding of both ubiquinol (QH 2 ) and the dissociated oxidized iron -sulfur protein (ISP ox ). A binding constant of f 14 can be estimated from the displacement of E m or pK for quinone or ISP ox , respectively. The binding likely involves a hydrogen bond, through which a proton-coupled electron transfer occurs. An enzyme -product complex is also formed at the Q o -site, in which ubiquinone (Q) hydrogen bonds with the reduced ISP (ISPH). The complex has been characterized in ESEEM experiments, which detect a histidine ligand, likely His-161 of ISP (in mitochondrial numbering), with a configuration similar to that in the complex of ISPH with stigmatellin. This special configuration is lost on binding of myxothiazol. Formation of the H-bond has been explored through the redox dependence of cytochrome c oxidation. We confirm previous reports of a decrease in E m of ISP on addition of myxothiazol, and show that this change can be detected kinetically. We suggest that the myxothiazol-induced change reflects loss of the interaction of ISPH with Q, and that the change in E m reflects a binding constant of f 4. We discuss previous data in the light of this new hypothesis, and suggest that the native structure might involve a less than optimal configuration that lowers the binding energy of complexes formed at the Q o -site so as to favor dissociation. We also discuss recent results from studies of the bypass reactions at the site, which lead to superoxide (SO) production under aerobic conditions, and provide additional information about intermediate states. D
Journal of Biological Chemistry, 2010
The rate-determining step in the overall turnover of the bc 1 complex is electron transfer from ubiquinol to the Rieske ironsulfur protein (ISP) at the Q o -site. Structures of the ISP from Rhodobacter sphaeroides show that serine 154 and tyrosine 156 form H-bonds to S-1 of the [2Fe-2S] cluster and to the sulfur atom of the cysteine liganding Fe-1 of the cluster, respectively. These are responsible in part for the high potential (E m,7 ϳ300 mV) and low pK a (7.6) of the ISP, which determine the overall reaction rate of the bc 1 complex. We have made site-directed mutations at these residues, measured thermodynamic properties using protein film voltammetry to evaluate the E m and pK a values of ISPs, explored the local proton environment through two-dimensional electron spin echo envelope modulation, and characterized function in strains S154T, S154C, S154A, Y156F, and Y156W. Alterations in reaction rate were investigated under conditions in which concentration of one substrate (ubiquinol or ISP ox ) was saturating and the other was varied, allowing calculation of kinetic terms and relative affinities. These studies confirm that H-bonds to the cluster or its ligands are important determinants of the electrochemical characteristics of the ISP, likely through electron affinity of the interacting atom and the geometry of the H-bonding neighborhood. The calculated parameters were used in a detailed Marcus-Brønsted analysis of the dependence of rate on driving force and pH. The proton-first-then-electron model proposed accounts naturally for the effects of mutation on the overall reaction. spin echo envelope modulation; EPPS, N-(2hydroxyethyl)piperazine-NЈ-3-propanesulfonic acid; H-bond, hydrogen bond; HYSCORE, hyperfine sublevel correlation spectroscopy; ISP, Rieske iron-sulfur protein; ISPH, reduced from of ISP at physiological pH; MES, 4-morpholineethanesulfonic acid; PT/ET, proton-first-then-electron; Q or quinone, ubiquinone; QH 2 , ubihydroquinone; Q i -site, site of bc 1 complex at which quinone is reduced; Q o -site, site of the bc 1 complex at which QH 2 is oxidized; MOPS, 4-morpholinepropanesulfonic acid; RC, photochemical reaction center; SQ, ubisemiquinone; TAPS, N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid; WT, wild type.
Biochemistry, 1995
One-and two-dimensional (1D and 2D) electron spin-echo envelope modulation (ESEEM) spectroscopy has been used to investigate the ligand environment of the [2Fe-2S] cluster from the terminal dioxygenase (ISPBED) of the Pseudomonas putida benzene dioxygenase complex. The modulation frequencies observed in the 0.5-8.5 MHz region of the Fourier transforms of 1D and 2D ESEEM spectra measured across the electron paramagnetic resonance (EPR) absorbance envelope (from g, through to gx) are consistent with their assignment to two I4N nuclei. Using hyperfine sublevel correlation spectroscopy (HYSCORE), two sets of correlated double quantum transitions sharing the same hyperfine coupling were observed and were identified as being due to the same two I4N nuclei. On the basis of the isotropic hyperfine and quadrupolar couplings estimated for these I4N nuclei [N(l), Aiso = 3.6 MHz and e2qQ = 2.2-2.8 MHz; N(2), Aiso = 4.8 MHz and e2qQ = 2.2-2.4 MHz], the ESEEM pattern of ISPBED is assigned to two histidine nitrogens which are directly coordinated to the reduced iron-sulfur cluster. Bonding parameters of the two [I4N]histidine ligands were calculated from these hyperfine couplings. The primary covalent contributions to the hyperfine interaction arise from I4N-to-Fe2+ u bonds. For N( l), our analysis of the percentage of unpaired 2s and 2p electrons gavefZs 1.3% andfzp -0.2%, while values offzs -1.7% andfzp -1.4% were estimated for N(2). Comparison of these values with those determined from electron nuclear double resonance (ENDOR) data of the Rieske-type [2Fe-2S] center of Pseudomonas cepacia phthalate dioxygenase Biochemistry 28, 4861-48711 indicates an apparent reduction in unpaired electron spin density residing on the two I4N ligands of ISPBED. Analysis of slices of the HYSCORE spectrum has provided evidence for another I4N nucleus (A -1.1 MHz, e2qQ = 3.3 MHz), which we have attributed to a weakly coupled peptide nitrogen, similar to those observed for ferredoxin-type [2Fe-2S] clusters. This type of weak interaction has not been previously described by the detailed ENDOR and ESEEM studies of Rieske-type centers. The resolution of the spectra demonstrates the effectiveness of 2D ESEEM for the disentanglement of multiple hyperfine interactions to metalloprotein centers.
Structure, 2007
The Rieske [2Fe-2S] iron-sulfur protein of cytochrome bc 1 functions as the initial electron acceptor in the rate-limiting step of the catalytic reaction. Prior studies have established roles for a number of conserved residues that hydrogen bond to ligands of the [2Fe-2S] cluster. We have constructed sitespecific variants at two of these residues, measured their thermodynamic and functional properties, and determined atomic resolution X-ray crystal structures, for the native protein at 1.2 Å resolution, and for five variants (Ser-154→Ala, Ser-154→Thr, Ser-154→Cys, Tyr-156→Phe and Tyr-156→Trp) to resolutions between 1.5 Å and 1.1 Å. These structures and complementary biophysical data provide a molecular framework for understanding the role of hydrogen bonds to the cluster in tuning thermodynamic properties, and hence the rate of this bioenergetic reaction. These studies provide the first detailed structure-function dissection of the role of hydrogen bonds in tuning the redox potentials of [2Fe-2S] clusters.
Journal of the American Chemical Society, 2011
Although the majority of noncovalent interactions associated with hydrogen and heavy atoms in proteins and other biomolecules are classical hydrogen bonds between polar N-H or O-H moieties and O atoms or aromatic π electrons, high-resolution X-ray crystallographic models deposited in the Protein Data Bank show evidence for weaker C-H 3 3 3 O hydrogen bonds, including ones involving sp 3 -hybridized carbon atoms. Little evidence is available in proteins for the (even) weaker C-H 3 3 3 S interactions described in the crystallographic literature on small molecules. Here, we report experimental evidence and theoretical verification for the existence of nine aliphatic (sp 3 -hybridized) C-H 3 3 3 S 3-center-4-electron interactions in the protein Clostridium pasteurianum rubredoxin. Our evidence comes from the analysis of carbon-13 NMR chemical shifts assigned to atoms near the iron at the active site of this protein. We detected anomalous chemical shifts for these carbon-13 nuclei and explained their origin in terms of unpaired spin density from the iron atom being delocalized through interactions of the type: C-H 3 3 3 S-Fe, where S is the sulfur of one of the four cysteine side chains covalently bonded to the iron. These results suggest that polarized sulfur atoms in proteins can engage in multiple weak interactions with surrounding aliphatic groups. We analyze the strength and angular dependence of these interactions and conclude that they may contribute small, but significant, stabilization to the molecule.
Physical Chemistry Chemical Physics, 2009
We have used X-band ESEEM to study the reduced [2Fe-2S] cluster in adrenodoxin and Arthrospira platensis ferredoxin. By use of a 2D approach (HYSCORE), we have shown that the cluster is involved in weak magnetic interactions with several nitrogens in each protein. Despite substantial difference in the shape and orientational dependence of individual crosspeaks, the major spectral features in both proteins are attributable to two peptide nitrogens (N1 and N2) with similar hyperfine couplings ∼1.1 and ∼0.70 MHz. The couplings determined represent to a small fraction (0.0003-0.0005) of the unpaired spin density of the reduced cluster transferred to these nitrogens over H-bond bridges or the covalent bonds of cysteine ligands. Simulation of the HYSCORE spectra has allowed us to estimate the orientation of the nuclear quadrupole tensors of N1 and N2 in the g-tensor coordinate system. The most likely candidates for the role of N1 and N2 have been identified in the protein environment by comparing magnetic-resonance data with crystallographic structures of the oxidized proteins. A possible influence of redox-linked structural changes on ESEEM data is analyzed using available structures for related proteins in two redox states. † Electronic supplementary information (ESI) available: The nuclear frequencies of an I=1 nucleus interacting with an electron spin S=1/2; an example of two-pulse ESEEM; direction cosines of the g-tensor principal axes in the coordinate system of the X-ray structure of two proteins; distances between bridging sulfurs and sulfurs of cysteine ligands and nearest protein nitrogens for two proteins; anisotropic hyperfine tensors calculated in the point dipole approximation for the nitrogens suitable for hydrogen bond formation with cluster sulfurs; rescaling of isotropic hyperfine couplings; structure around the cluster in two proteins.
Exploration of Ligands to the Qi Site Semiquinone in the bc1 Complex Using High-resolution EPR
Journal of Biological Chemistry, 2003
Pulsed EPR spectroscopy was used to explore the structural neighborhood of the semiquinone (SQ) stabilized at the Q i site of the bc 1 complex of Rhodobacter sphaeroides (EC 1.10.2.2) and to demonstrate that the nitrogen atom of a histidine imidazole group donates an H-bond to the SQ. Crystallographic structures show two different configurations for the binding of ubiquinone at the Q i site of mitochondrial bc 1 complexes in which histidine (His-201 in bovine sequence) is either a direct H-bond donor or separated by a bridging water. The paramagnetic properties of the SQ formed at the site provide an independent method for studying the liganding of this intermediate species. The antimycin-sensitive SQ formed at the Q i site by either equilibrium redox titration, reduction of the oxidized complex by ascorbate, or addition of decylubihydroquinone to the oxidized complex in the presence of myxothiazol all showed similar properties. The electron spin echo envelope modulation spectra in the 14 N region were dominated by lines with frequencies at 1.7 and 3.1 MHz. Hyperfine sublevel correlation spectroscopy spectra showed that these were contributed by a single nitrogen. Further analysis showed that the 14 N nucleus was characterized by an isotropic hyperfine coupling of ϳ0.8 MHz and a quadrupole coupling constant of ϳ0.35 MHz. The nitrogen was identified as the N-⑀ or N-␦ imidazole nitrogen of a histidine (it is likely to be His-217, or His-201 in bovine sequence). A distance of 2.5-3.1 Å for the O-N distance between the carbonyl of SQ and the nitrogen was estimated. The mechanistic significance is discussed in the context of a dynamic role for the movement of His-217 in proton transfer to the site. who was an inspiration to all who worked in the field of advanced EPR and the areas of photosynthesis and biological electron transfer. Arnold was not only at the forefront of these fields but was also highly innovative in the development of new techniques and applications to research in this area. He will be sorely missed.