Parameters of quadrupole coupling of 14N nuclei in chlorophyll a cations determined by the electron spin echo method

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.The bc 1 complex family of enzymes plays a central role in all the main pathways of energy conversion, being directly responsible for ϳ30% of all the energy transduction of the biosphere (1-3). The complexes in Rhodobacter exemplify the simplest of these enzymes, with only three or four subunits, including the highly conserved catalytic core common to bacterial and mitochondrial complexes. It is generally accepted that the complex operates through a protonmotive Q cycle (2, 4, 5). Three catalytic subunits, cytochrome b, cytochrome c 1 , and the Rieske iron-sulfur protein, house the mechanism. Two separate internal electron transfer chains connect three catalytic sites for external substrates. At one site, cytochrome c 1 is oxidized by cytochrome c 2 . Two catalytic sites in cytochrome b are involved in the oxidation or reduction of ubiquinone. At the quinol oxidizing site, one electron from quinol is passed to the ironsulfur protein, which transfers it to cytochrome c 1 , whereas the semiquinone (SQ) 1 produced is oxidized by another chain consisting of the two b-hemes of cytochrome b in the bifurcated reaction. At the quinone-reducing site (Q i site), electrons from the b-heme chain are used to generate quinol. The integration of the oxidation and reduction reactions with the release or uptake of protons in the aqueous pha...

Section: Resultsmentioning

confidence: 99%

Exploration of Ligands to the Qi Site Semiquinone in the bc 1 Complex Using High-resolution EPR

Kolling

Samoilova

Holland

et al. 2003

“…In measurements of amorphous (powder) samples, such as the frozen suspensions of cytochrome bo 3 used in this work, because of their different orientation dependence, not all transitions contribute equally to the spectra. The type of spectrum expected from 14 N with predominantly isotropic hyperfine coupling A is governed by the ratio between the effective nuclear frequency in each manifold, efϮ , given by efϮ ϭ ͉ N Ϯ ͉A͉/2͉ ( N is the Zeeman frequency of 14 N), and the quadrupole coupling constant, K, given by K ϭ e 2 qQ/4h (21,22). If efϮ /K ϳ 0, i.e.…”

Section: Methodsmentioning

confidence: 99%

Characterization of Mutants That Change the Hydrogen Bonding of the Semiquinone Radical at the QH Site of the Cytochrome bo3 from Escherichia coli

Yap

Samoilova

Gennis

et al. 2007

The cytochrome bo 3 ubiquinol oxidase catalyzes the two-electron oxidation of ubiquinol in the cytoplasmic membrane of Escherichia coli, and reduces O 2 to water. This enzyme has a high affinity quinone binding site (Q H ), and the quinone bound to this site acts as a cofactor, necessary for rapid electron transfer from substrate ubiquinol, which binds at a separate site (Q L ), to heme b. Previous pulsed EPR studies have shown that a semiquinone at the Q H site formed during the catalytic cycle is a neutral species, with two strong hydrogen bonds to Asp-75 and either Arg-71 or Gln-101. In the current work, pulsed EPR studies have been extended to two mutants at the Q H site. The D75E mutation has little influence on the catalytic activity, and the pattern of hydrogen bonding is similar to the wild type. In contrast, the D75H mutant is virtually inactive. Pulsed EPR revealed significant structural changes in this mutant. The hydrogen bond to Arg-71 or Gln-101 that is present in both the wild type and D75E mutant oxidases is missing in the D75H mutant. Instead, the D75H has a single, strong hydrogen bond to a histidine, likely His-75. The D75H mutant stabilizes an anionic form of the semiquinone as a result of the altered hydrogen bond network. Either the redistribution of charge density in the semiquinone species, or the altered hydrogen bonding network is responsible for the loss of catalytic function. Escherichia coli cytochrome (cyt)3 bo 3 ubiquinol oxidase catalyzes the two-electron oxidation of ubiquinol and the fourelectron reduction of O 2 to water. The enzyme contains three redox-active metal centers: a low spin heme b, which is involved in quinol oxidation, and the heme o 3 /Cu B bimetallic center, which is the site where O 2 binds and is reduced to water. The ubiquinol oxidation occurs with a semiquinone (SQ) intermediate in an overall reaction that releases two protons to the periplasm. The enzyme contains two Q sites (1-6): a low affinity site (Q L ), which is equilibrated with the quinone pool in the membrane and functions as the substrate (QH 2 ) binding site, and a high affinity (Q H ) site, from which Q is not readily removed, and that stabilizes a SQ (7-10). The quinone bound at the high-affinity site appears to function as a tightly bound cofactor, similar to the Q A site of the reaction centers. Rapid kinetic studies show that the quinone bound at the Q H site is important for rapid reduction of heme b but not for rapid electron transfer from heme b to the heme o 3 /Cu B binuclear center (11,12). The heme o 3 /Cu B site is where O 2 is reduced to H 2 O using the electrons provided by the oxidation of quinol. Hence, the suggested electron transfer sequence is as follows in Reaction 1.The x-ray structure of cytochrome bo 3 (13) does not contain any bound quinone, but site-directed mutagenesis studies (2-4, 13) have identified residues that modulate the properties of the Q H site. The model of the Q H quinone binding site (Fig. 1), including Arg-71, Asp-75, His-98, and Gln-101 residues (13), has...

“…In measurements of amorphous (powder) samples (such as the frozen suspensions of the ISF used here), because of their different orientation dependence, not all transitions contribute equally to the spectra. The type of spectrum expected from 14 N with predominantly isotropic hyperfine coupling A is governed by the ratio between the effective nuclear frequency in each manifold, efϮ , given by efϮ ϭ ͉ I Ϯ ͉A͉/2͉, and the quadrupole coupling constant, K, given by K ϭ e 2 qQ/4h (23,24). If efϮ /K ϭ ϳ0, i.e.…”

Section: Methodsmentioning

confidence: 99%

“…Further studies of the Rieske cluster by X-band oneand two-dimensional ESEEM (20 -22) have shown that the major contribution in these spectra comes from the coordinating N ␦ of histidines. Other more weakly coupled 14 N nitrogens present in the cluster environment do not produce recognizable lines in the spectra, because of the influence of the nuclear quadrupole interaction of 14 N. Whether or not lines are seen depends on a particular relation between nuclear Zeeman frequency and hyperfine coupling (23,24), as explained under "Experimental Procedures." Recently, it was demonstrated that the weakly coupled nitrogens produce readily observed lines in X-band two-dimensional ESEEM (HYSCORE) spectra of 15 Nlabeled sulredoxin, another Rieske-like protein (25).…”

mentioning

confidence: 98%

Identification of Hydrogen Bonds to the Rieske Cluster through the Weakly Coupled Nitrogens Detected by Electron Spin Echo Envelope Modulation Spectroscopy

Dikanov

Kolling

Endeward

et al. 2006

Proteins containing Rieske-type [2Fe-2S] clusters with two histidyl and two cysteinyl ligands play important roles in many biological electron transfer reactions such as aerobic respiration, photosynthesis, and biodegradation of various alkene and aromatic compounds. The distinct biological functions of this protein family are in part associated with the cluster redox potential and the pK of the oxidized form, which are roughly correlated with the number of hydrogen bonds from protein side chains and the peptide backbone to the cluster and its immediate ligands (1-6).In the cytochrome bc 1 /b 6f family, a Rieske iron-sulfur protein (ISP) 4 is a constituent of the high potential electron transfer chain that accepts the first electron in the bifurcated reaction at the ubihydroquinone (quinol, QH 2 ) oxidizing Q o site. In the catalytic mechanism at the Q o site, the redox reaction involves extraction of both an electron and a proton from the bound quinol and their transfer to the ISP through a short pathway that includes the ISP His-161 ring and the H-bond between N ⑀ and the -OH of the quinol substrate (7). It has been proposed that the electron transfer is gated by the low probability of finding the H-atom of the H-bond in the kinetically favorable position, determined by the pK difference between the quinol and the oxidized ISP (ISP ox ) (reviewed in Refs. 5 and 6). The pK on ISP ox is contributed by one of the cluster ligands, His-161 in bovine numbering, and is associated with H ϩ dissociation from the N ⑀ involved in the H-bond. The gating accounts for the fact that this first electron transfer is slower by more than 3 orders of magnitude than would be expected from the short distance (ϳ7 Å) involved (6, 7). The first electron transfer is the ratedetermining step in quinol oxidation under conditions of substrate saturation. Rate determination at this reaction is demonstrated by the fact that the overall rate depends on its driving force in a classical Marcus fashion, as shown through use of mutants of the ISP with modified redox potential (6 -13). Briefly, the driving force, ⌬G o , for the first electron transfer is determined by the redox potential difference between the donor (SQ/QH 2 ) and acceptor (ISP ox /ISPH) couples. Assuming that mutations in the ISP do not change E m (SQ/QH 2 ), a change in * This work was supported by National Institutes of Health Grants GM 35438 (to A. R. C.) and GM 62954 (to S. A. D.), Fogarty Grant PHS 1 RO3 TW 01495 (to A. R. C. and R. I. S.), and National Institutes of Health/National Center for Research Resources Grant S10-RR15878 for instrumentation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 To whom correspondence may be addressed. E-mail: dikanov@uiuc.edu. 2 Present address: Dept. of Chemistry, Princeton University, Princeton, NJ 08540. 3 To whom correspondence may be addressed. E-mail:...