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

Dikanov, Sergei A.; Kolling, Derrick R.J.; Endeward, Burkhard; Samoilova, Rimma I.; Prisner, Thomas F.; Nair, Satish K.; Crofts, Antony R.

doi:10.1074/jbc.m604103200

Cited by 27 publications

(66 citation statements)

References 53 publications

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“…These have been studied in detail through mutagenesis [13, 59, 64, 65], thermodynamic characterization [13, 62], spectroscopic studies [66–68], crystallographic structures at atomic resolution [11, 13], and detailed kinetic analysis [13, 44, 47, 51]. Complementary work using NMR and specific isotopic labeling in Thermus thermophilus [69, 70] has identified the group responsible for the low p K ox1 in ISP as the histidine equivalent to His-152 in Rb.…”

Section: The Qo-site Reactionmentioning

confidence: 99%

The mechanism of ubihydroquinone oxidation at the Qo-site of the cytochrome bc1 complex

Crofts

Hong

Wilson

et al. 2013

Biochimica et Biophysica Acta (BBA) - Bioenergetics

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117

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1. Recent results suggest that the major flux is carried by a monomeric function, not by intermonomer electron flow. 2. The bifurcated reaction at the Qo-site involves sequential partial processes, - a rate limiting first electron transfer generating a semiquinone (SQ) intermediate, and a rapid second electron transfer in which the SQ is oxidized by the low potential chain. 3. The rate constant for the first step in a strongly endergonic, proton-first-then-electron mechanism, is given by a Marcus-Brønsted treatment in which a rapid electron transfer is convoluted with a weak occupancy of the proton configuration needed for electron transfer. 4. A rapid second electron transfer pulls the overall reaction over. Mutation of Glu-295 of cyt b shows it to be a key player. 5. In more crippled mutants, electron transfer is severely inhibited and the bell-shaped pH dependence of wildtype is replaced by a dependence on a single pK at ~8.5 favoring electron transfer. Loss of a pK ~6.5 is explained by a change in the rate limiting step from the first to the second electron transfer; the pK ~8.5 may reflect dissociation of QH·. 6. A rate constant (<103 s−1) for oxidation of SQ in the distal domain by heme bL has been determined, which precludes mechanisms for normal flux in which SQ is constrained there. 7. Glu-295 catalyzes proton exit through H+ transfer from QH·, and rotational displacement to delivers the H+ to exit channel(s). This opens a volume into which Q·− can move closer to the heme to speed electron transfer. 8. A kinetic model accounts well for the observations, but leaves open the question of gating mechanisms. For the first step we suggest a molecular “escapement”; for the second a molecular ballet choreographed through coulombic interactions.

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Section: The Qo-site Reactionmentioning

confidence: 99%

The mechanism of ubihydroquinone oxidation at the Qo-site of the cytochrome bc1 complex

Crofts

Hong

Wilson

et al. 2013

Biochimica et Biophysica Acta (BBA) - Bioenergetics

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117

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“…Two different preparations of the ISP were used, a proteolyzed iron-sulfur protein fragment, used in spectroscopic (35) and structural studies (8), and an intact subunit, used for protein film voltammetry because it adhered better to the graphite electrode (33). The subunit from the mutant bc 1 complexes was FIGURE 1.…”

Section: Methodsmentioning

confidence: 99%

“…This indicates that the mutations do not influence the geometry of histidine coordination. However, weakly coupled interactions (for example through H-bonding) are not well resolved by 14 N ESEEM at X-band, and more extensive studies using 15 N and S-band might reveal changes in the nitrogen environment of the cluster (35).…”

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confidence: 99%

Modifications of Protein Environment of the [2Fe-2S] Cluster of the bc1 Complex

Lhee

Kolling

Nair

et al. 2010

Journal of Biological Chemistry

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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.Enzymes of the cytochrome bc 1 complex 2 family (EC 1.10.2.2) are ubiquitous membrane proteins in mitochondria and bacteria (and, including the b 6 f complex, in chloroplasts) that play a central role in the electron transfer chains of respiration and photosynthesis. In Rhodobacter sphaeroides, the bc 1 complex, together with the reaction center (RC) and cytochrome (cyt) c 2 , forms the photosynthetic chain. The complex catalyzes oxidation of substrate ubiquinol (QH 2 ) by a watersoluble ferricytochrome c 2 in the periplasmic space and generates a proton-motive force for ATP synthesis through a Q-cycle mechanism (1, 2). With the availability of structures from mitochondrial and bacterial systems (3-8), the mechanism could be further refined. Proteins of the bc 1 complex family form homodimers inserted in the cellular or mitochondrial membrane, each monomer containing minimally the three subunits (cyt b with two b-type hemes (b H and b L ), cyt c 1 with heme c 1 , and Rieske-type iron-sulfur protein (ISP)) that form the catalytic core of the complex. The ␣-proteobacterial ancestors of mitochondria were likely derived from photosynthetic progenitors, accounting for the high degree of homology in sequence, structure, and mechanism. The bacterial structures are much simpler than the 8 -11 subunit complexes in mitochondria. The isolated complex has four subunits in each monomer (cyt b, cyt c 1 , ISP, and subunit IV) (9, 10), but crystallo...

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“…The concentration of chromatophores was adjusted to give an absorbance of ⌬A 562-577 nm ϭ 1.2, and solubilization was performed for 1 h. Unless otherwise indicated, the bc 1 complex was used without added cryoprotectants, because Me 2 SO or glycerol decreased the amplitude of the Q i -site semiquinone signal. Protein isotopically labeled with 15 N was prepared from R. sphaeroides grown in Sistrom media as described previously (36,37).…”

Section: Methodsmentioning

confidence: 99%

Hydrogen Bonds between Nitrogen Donors and the Semiquinone in the Qi-site of the bc1 Complex

Dikanov

Holland

Endeward

et al. 2007

Journal of Biological Chemistry

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The ubisemiquinone stabilized at the Q i -site of the bc 1 complex of Rhodobacter sphaeroides forms a hydrogen bond with a nitrogen from the local protein environment, tentatively identified as ring N from His-217. The interactions of 14 N and 15 N have been studied by X-band (ϳ9.7 GHz) and S-band (3.4 GHz) pulsed EPR spectroscopy. The application of S-band spectroscopy has allowed us to determine the complete nuclear quadrupole tensor of the 14 N involved in H-bond formation and to assign it unambiguously to the N ⑀ of His-217. This tensor has distinct characteristics in comparison with H-bonds between semiquinones and N ␦ in other quinone-processing sites. The experiments with 15 N showed that the N ⑀ of His-217 was the only nitrogen carrying any considerable unpaired spin density in the ubiquinone environment, and allowed calculation of the isotropic and anisotropic couplings with the N ⑀ of His-217. From these data, we could estimate the unpaired spin density transferred onto 2s and 2p orbitals of nitrogen and the distance from the nitrogen to the carbonyl oxygen of 2.38 ؎ 0.13 Å . The hyperfine coupling of other protein nitrogens with semiquinone is <0.1 MHz. This did not exclude the nitrogen of the Asn-221 as a possible hydrogen bond donor to the methoxy oxygen of the semiquinone. A mechanistic role for this residue is supported by kinetic experiments with mutant strains N221T, N221H, N221I, N221S, N221P, and N221D, all of which showed some inhibition but retained partial turnover.The bc 1 complex (ubihydroquinone:cytochrome c oxidoreductase, or complex III of the mitochondrial respiratory chain) functions to oxidize ubihydroquinone (QH 2 , 4 quinol) in the membrane and reduce cytochrome c (or cytochrome c 2 in bacteria) in the aqueous phase, using the redox work to transport 2H ϩ per electron across the membrane (1-4). In the photosynthetic bacterium Rhodobacter sphaeroides, the bc 1 complex completes the photosynthetic chain containing the photochemical reaction center, which provides the substrates for the complex following photoactivation (5, 6). The Q-cycle mechanism is a well tested model to account for the function of the bc 1 complex (7-9), but several details are still controversial. The Q-cycle involves separate catalytic sites on opposite sides of the membrane for oxidation of QH 2 and reduction of ubiquinone (Q, quinone). The work function is provided by a bifurcation of electron transfer at the quinol-oxidizing site (the Q osite). The first electron is transferred to a high potential chain (the Rieske iron-sulfur protein, cytochrome c 1 , and cytochrome c), and the second to a low potential chain, consisting of heme b L and heme b H in cytochrome b, which delivers electrons to the quinone reduction site (Q i -site). Some of the work from the first electron transfer is stored in the strong reducing potential of an unstable semiquinone intermediate formed at the Q o -site. This provides the driving force for reduction of Q, or of ubisemiquinone (SQ), in the Q i -site, and for generation of the p...

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

Cited by 27 publications

References 53 publications

The mechanism of ubihydroquinone oxidation at the Qo-site of the cytochrome bc1 complex

The mechanism of ubihydroquinone oxidation at the Qo-site of the cytochrome bc1 complex

Modifications of Protein Environment of the [2Fe-2S] Cluster of the bc1 Complex

Hydrogen Bonds between Nitrogen Donors and the Semiquinone in the Qi-site of the bc1 Complex

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