The modified Q-cycle: A look back at its development and forward to a functional model.

On looking back at a lifetime of research, it is interesting to see, in the light of current progress, how things came to be, and to speculate on how things might be. I am delighted in the context of the Mitchell prize to have that excuse to present this necessarily personal view of developments in areas of my interests. I have focused on the Q-cycle and a few examples showing wider ramifications, since that had been the main interest of the lab in the 20 years since structures became available, - a watershed event in determining our molecular perspective. I have reviewed the evidence for our model for the mechanism of the first electron transfer of the bifurcated reaction at the Qo-site, which I think is compelling. In reviewing progress in understanding the second electron transfer, I have revisited some controversies to justify important conclusions which appear, from the literature, not to have been taken seriously. I hope this does not come over as nitpicking. The conclusions are important to the final section in which I develop an internally consistent mechanism for turnovers of the complex leading to a state similar to that observed in recent rapid-mix/freeze-quench experiments, reported three years ago. The final model is necessarily speculative but is open to test.

Dissecting the pattern of proton release from partial process involved in ubihydroquinone oxidation in the Q-cycle.

A key feature of the modified Q-cycle of the cytochrome bc1 and related complexes is a bifurcation of QH2 oxidation involving electron transfer to two different acceptor chains, each coupled to proton release. We have studied the kinetics of proton release in chromatophore vesicles from Rhodobacter sphaeroides, using the pH-sensitive dye neutral red to follow pH changes inside on activation of the photosynthetic chain, focusing on the bifurcated reaction, in which 4H+are released on complete turnover of the Q-cycle (2H+/ubiquinol (QH2) oxidized). We identified different partial processes of the Qo-site reaction, isolated through use of specific inhibitors, and correlated proton release with electron transfer processes by spectrophotometric measurement of cytochromes or electrochromic response. In the presence of myxothiazol or azoxystrobin, the proton release observed reflected oxidation of the Rieske iron­sulfur protein. In the absence of Qo-site inhibitors, the pH change measured represented the convolution of this proton release with release of protons on turnover of the Qo-site, involving formation of the ES-complex and oxidation of the semiquinone intermediate. Turnover also regenerated the reduced iron-sulfur protein, available for further oxidation on a second turnover. Proton release was well-matched with the rate limiting step on oxidation of QH2 on both turnovers. However, a minor lag in proton release found at pH 7 but not at pH 8 might suggest that a process linked to rapid proton release on oxidation of the intermediate semiquinone involves a group with a pK in that range.

The Q-Cycle Mechanism of the bc1 Complex: A Biologist's Perspective on Atomistic Studies.

The Q-cycle mechanism of the bc1 complex is now well enough understood to allow application of advanced computational approaches to the study of atomistic processes. In addition to the main features of the mechanism, these include control and gating of the bifurcated reaction at the Qo-site, through which generation of damaging reactive oxygen species is minimized. We report a new molecular dynamics model of the Rhodobacter sphaeroides bc1 complex implemented in a native membrane, and constructed so as to eliminate blemishes apparent in earlier Rhodobacter models. Unconstrained MD simulations after equilibration with ubiquinol and ubiquinone respectively at Qo- and Qi-sites show that substrate binding configurations at both sites are different in important details from earlier models. We also demonstrate a new Qo-site intermediate, formed in the sub-ms time range, in which semiquinone remains complexed with the reduced iron sulfur protein. We discuss this, and a spring-loaded mechanism for modulating interactions of the iron sulfur protein with occupants of the Qo-site, in the context of control and gating roles. Such atomistic features of the mechanism can usefully be explored through simulation, but we stress the importance of constraints from physical chemistry and biology, both in setting up a simulation and in interpreting results.

New functional and biophysical insights into the mitochondrial Rieske iron-sulfur protein from genetic suppressor analysis in C. elegans.

Several intragenic mutations suppress the C. elegans isp-1(qm150) allele of the mitochondrial Rieske iron-sulfur protein (ISP), a catalytic subunit of Complex III of the respiratory chain. These mutations were located in a helical region of the "tether" span of ISP-1, distant from the primary mutation in the extrinsic head, and suppressed all pleiotropic phenotypes associated with the qm150 allele. Analysis of these suppressors revealed control of electron transfer into Complex III through a "spring-loaded" mechanism involving a binding force for formation of enzyme-substrate complex, counter balanced by forces (a chemical "spring") favoring helix formation in the tether. The primary P→S mutation results in inhibition of electron flow into the Q-cycle by decreasing the binding force, and the tether mutations relieve this inhibition by weakening the "spring." In this commentary we discuss additional control features, and relate the primary inhibition to outcomes at the organismal level. In particular, the sensitivity to hyperoxia and the elevated reactive oxygen species (ROS) seen in isp-1(qm150), likely reflect over-reduction of the quinone pool, which is upstream of the inhibited site; at high O2, this would lead to increased ROS production through complex I. We speculate that alternative NADH:ubiquinone oxidoreductase activity in C. elegans from the worm apoptosis inducing factor (AIF) homolog (WAH-1) might also be involved, and that WAH-1 might have a "canary" function in detection of this adverse state (high O2/reduced pool), and a role in protection of the organism by transformation to AIF-like products, and apoptotic recycling of defective cells.

In photosynthesis, oxygen comes from water: from a 1787 book for women by Monsieur De Fourcroy.

It is now well established that the source of oxygen in photosynthesis is water. The earliest suggestion previously known to us had come from René Bernard Wurmser (1930). Here, we highlight an earlier report by Monsieur De Fourcroy (1787), who had already discussed the broad outlines of such a hypothesis in a book on Chemistry written for women. We present here a free translation of a passage from this book, with the original text in French as an Appendix.

Tether mutations that restore function and suppress pleiotropic phenotypes of the C. elegans isp-1(qm150) Rieske iron-sulfur protein.

Mitochondria play an important role in numerous diseases as well as normative aging. Severe reduction in mitochondrial function contributes to childhood disorders such as Leigh Syndrome, whereas mild disruption can extend the lifespan of model organisms. The Caenorhabditis elegans isp-1 gene encodes the Rieske iron-sulfur protein subunit of cytochrome c oxidoreductase (complex III of the electron transport chain). The partial loss of function allele, isp-1(qm150), leads to several pleiotropic phenotypes. To better understand the molecular mechanisms of ISP-1 function, we sought to identify genetic suppressors of the delayed development of isp-1(qm150) animals. Here we report a series of intragenic suppressors, all located within a highly conserved six amino acid tether region of ISP-1. These intragenic mutations suppress all of the evaluated isp-1(qm150) phenotypes, including developmental rate, pharyngeal pumping rate, brood size, body movement, activation of the mitochondrial unfolded protein response reporter, CO2 production, mitochondrial oxidative phosphorylation, and lifespan extension. Furthermore, analogous mutations show a similar effect when engineered into the budding yeast Rieske iron-sulfur protein Rip1, revealing remarkable conservation of the structure-function relationship of these residues across highly divergent species. The focus on a single subunit as causal both in generation and in suppression of diverse pleiotropic phenotypes points to a common underlying molecular mechanism, for which we propose a "spring-loaded" model. These observations provide insights into how gating and control processes influence the function of ISP-1 in mediating pleiotropic phenotypes including developmental rate, movement, sensitivity to stress, and longevity.

Identification of ubiquinol binding motifs at the Qo-site of the cytochrome bc1 complex.

Enzymes of the bc1 complex family power the biosphere through their central role in respiration and photosynthesis. These enzymes couple the oxidation of quinol molecules by cytochrome c to the transfer of protons across the membrane, to generate a proton-motive force that drives ATP synthesis. Key for the function of the bc1 complex is the initial redox process that involves a bifurcated electron transfer in which the two electrons from a quinol substrate are passed to different electron acceptors in the bc1 complex. The electron transfer is coupled to proton transfer. The overall mechanism of quinol oxidation by the bc1 complex is well enough characterized to allow exploration at the atomistic level, but details are still highly controversial. The controversy stems from the uncertain binding motifs of quinol at the so-called Qo active site of the bc1 complex. Here we employ a combination of classical all atom molecular dynamics and quantum chemical calculations to reveal the binding modes of quinol at the Qo-site of the bc1 complex from Rhodobacter capsulatus. The calculations suggest a novel configuration of amino acid residues responsible for quinol binding and support a mechanism for proton-coupled electron transfer from quinol to iron-sulfur cluster through a bridging hydrogen bond from histidine that stabilizes the reaction complex.

The semiquinone at the Qi site of the bc1 complex explored using HYSCORE spectroscopy and specific isotopic labeling of ubiquinone in Rhodobacter sphaeroides via (13)C methionine and construction of a methionine auxotroph.

Specific isotopic labeling at the residue or substituent level extends the scope of different spectroscopic approaches to the atomistic level. Here we describe (13)C isotopic labeling of the methyl and methoxy ring substituents of ubiquinone, achieved through construction of a methionine auxotroph in Rhodobacter sphaeroides strain BC17 supplemented with l-methionine with the side chain methyl group (13)C-labeled. Two-dimensional electron spin echo envelope modulation (HYSCORE) was applied to study the (13)C methyl and methoxy hyperfine couplings in the semiquinone generated in situ at the Qi site of the bc1 complex in its membrane environment. The data were used to characterize the distribution of unpaired spin density and the conformations of the methoxy substituents based on density functional theory calculations of (13)C hyperfine tensors in the semiquinone of the geometry-optimized X-ray structure of the bc1 complex (Protein Data Bank entry 1PP9 ) with the highest available resolution. Comparison with other proteins indicates individual orientations of the methoxy groups in each particular case are always different from the methoxy conformations in the anion radical prepared in a frozen alcohol solution. The protocol used in the generation of the methionine auxotroph is more generally applicable and, because it introduces a gene deletion using a suicide plasmid, can be applied repeatedly.

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

1. Recent results suggest that the major flux is carried by a monomeric function, not by an 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 (<10(3)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 deliver 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. This article is part of a Special Issue entitled: Respiratory complex III and related bc complexes.

Role of the -PEWY-glutamate in catalysis at the Q(o)-site of the Cyt bc(1) complex.

We re-examine the pH dependence of partial processes of ubihydroquinone (QH(2)) turnover in Glu-295 mutants in Rhodobacter sphaeroides to clarify the mechanistic role. In more crippled mutants, the bell-shaped pH profile of wildtype was replaced by dependence on a single pK at ~8.5 favoring electron transfer. Loss of the pK at 6.5 reflects a change in the rate-limiting step from the first to the second electron transfer. Over the range of pH 6-8, no major pH dependence of formation of the initial reaction complex was seen, and the rates of bypass reactions were similar to the wildtype. Occupancy of the Q(o)-site by semiquinone (SQ) was similar in the wildtype and the Glu→Trp mutant. Since heme b(L) is initially oxidized in the latter, the bifurcated reaction can still occur, allowing estimation of an empirical rate constant <10(3)s(-1) for reduction of heme b(L) by SQ from the domain distal from heme b(L), a value 1000-fold smaller than that expected from distance. If the pK ~8.5 in mutant strains is due to deprotonation of the neutral semiquinone, with Q(•-) as electron donor to heme b(L), then in wildtype this low value would preclude mechanisms for normal flux in which semiquinone is constrained to this domain. A kinetic model in which Glu-295 catalyzes H(+) transfer from QH•, and delivery of the H(+) to exit channel(s) by rotational displacement, and facilitates rapid electron transfer from SQ to heme b(L) by allowing Q(•-) to move closer to the heme, accounts well for the observations.

Inter-monomer electron transfer is too slow to compete with monomeric turnover in bc(1) complex.

The homodimeric bc(1) complexes are membrane proteins essential in respiration and photosynthesis. The ~11Å distance between the two b(L)-hemes of the dimer opens the possibility of electron transfer between them, but contradictory reports make such inter-monomer electron transfer controversial. We have constructed in Rhodobacter sphaeroides a heterodimeric expression system similar to those used before, in which the bc(1) complex can be mutated differentially in the two copies of cyt b to test for inter-monomer electron transfer, but found that genetic recombination by cross-over then occurs to produce wild-type homodimer. Selection pressure under photosynthetic growth always favored the homodimer over heterodimeric variants enforcing inter-monomer electron transfer, showing that the latter are not competitive. These results, together with kinetic analysis of myxothiazol titrations, demonstrate that inter-monomer electron transfer does not occur at rates competitive with monomeric turnover. We examine the results from other groups interpreted as demonstrating rapid inter-monomer electron transfer, conclude that similar mechanisms are likely to be in play, and suggest that such claims might need to be re-examined.

Reaction of superoxide radical with quinone molecules.

When the superoxide radical O(2)(•-) is generated on reaction of KO(2) with water in dimethyl sulfoxide, the decay of the radical is dramatically accelerated by inclusion of quinones in the reaction mix. For quinones with no or short hydrophobic tails, the radical product is a semiquinone at much lower yield, likely indicating reduction of quinone by superoxide and loss of most of the semiquinone product by disproportionation. In the presence of ubiquinone-10, a different species (I) is generated, which has the EPR spectrum of superoxide radical. However, pulsed EPR shows spin interaction with protons in fully deuterated solvent, indicating close proximity to the ubinquinone-10. We discuss the nature of species I, and possible roles in the physiological reactions through which ubisemiquinone generates superoxide by reduction of O(2) through bypass reactions in electron transfer chains.

Modifications of protein environment of the [2Fe-2S] cluster of the bc1 complex: effects on the biophysical properties of the rieske iron-sulfur protein and on the kinetics of the complex.

The rate-determining step in the overall turnover of the bc(1) complex is electron transfer from ubiquinol to the Rieske iron-sulfur 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) approximately 300 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.

The reduced [2Fe-2S] clusters in adrenodoxin and Arthrospira platensis ferredoxin share spin density with protein nitrogens, probed using 2D ESEEM.

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 differences in the shape and orientational dependence of individual cross-peaks, the major spectral features in both proteins are attributable to two peptide nitrogens (N1 and N2) with similar hyperfine couplings approximately 1.1 and approximately 0.70 MHz. The couplings determined represent 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.

Proton environment of reduced Rieske iron-sulfur cluster probed by two-dimensional ESEEM spectroscopy.

The proton environment of the reduced [2Fe-2S] cluster in the water-soluble head domain of the Rieske iron-sulfur protein (ISF) from the cytochrome bc(1) complex of Rhodobacter sphaeroides has been studied by orientation-selected X-band 2D ESEEM. The 2D spectra show multiple cross-peaks from protons, with considerable overlap. Samples in which (1)H(2)O water was replaced by (2)H(2)O were used to determine which of the observed peaks belong to exchangeable protons, likely involved in hydrogen bonds in the neighborhood of the cluster. By correlating the cross-peaks from 2D spectra recorded at different parts of the EPR spectrum, lines from nine distinct proton signals were identified. Assignment of the proton signals was based on a point-dipole model for interaction with electrons of Fe(III) and Fe(II) ions, using the high-resolution structure of ISF from Rb. sphaeroides. Analysis of experimental and calculated tensors has led us to conclude that even 2D spectra do not completely resolve all contributions from nearby protons. Particularly, the seven resolved signals from nonexchangeable protons could be produced by at least 13 protons. The contributions from exchangeable protons were resolved by difference spectra ((1)H(2)O minus (2)H(2)O), and assigned to two groups of protons with distinct anisotropic hyperfine values. The largest measured coupling exceeded any calculated value. This discrepancy could result from limitations of the point dipole approximation in dealing with the distribution of spin density over the sulfur atoms of the cluster and the cysteine ligands, or from differences between the structure in solution and the crystallographic structure. The approach demonstrated here provides a paradigm for a wide range of studies in which hydrogen-bonding interactions with metallic centers has a crucial role in understanding the function.

D1-arginine257 mutants (R257E, K, and Q) of Chlamydomonas reinhardtii have a lowered QB redox potential: analysis of thermoluminescence and fluorescence measurements.

Arginine257 (R257), in the de-helix that caps the Q(B) site of the D1 protein, has been shown by mutational studies to play a key role in the sensitivity of Photosystem II (PS II) to bicarbonate-reversible binding of the formate anion. In this article, the role of this residue has been further investigated through D1 mutations (R257E, R257Q, and R257K) in Chlamydomonas reinhardtii. We have investigated the activity of the Q(B) site by studying differences from wild type on the steady-state turnover of PS II, as assayed through chlorophyll (Chl) a fluorescence yield decay after flash excitation. The effects of p-benzoquinone (BQ, which oxidizes reduced Q(B), Q(B)(-) ) and 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU, which blocks electron flow from Q(A)(-) to Q(B)) were measured. The equilibrium constants of the two-electron gate were obtained through thermoluminescence measurements. The thermoluminescence properties were changed in the mutants, especially when observed after pretreatment with 100 microM BQ. A theoretical analysis of the thermoluminescence data, based mainly on the recombination pathways model of Rappaport et al. (2005), led to the conclusion that the free-energy difference for the recombination of Q(B)(-) with S(2) was reduced by 20-40 mV in the three mutants (D1-R257K, D1-R257Q, and D1-R257E); this was interpreted to be due to a lowering of the redox potential of Q(B)/Q(B)(-). Further, since the recombination of Q(A)(-) with S(2) was unaffected, we suggest that no significant change in redox potential of Q(A)/Q(A)(-) occurred in these three mutants. The maximum variable Chl a fluorescence yield is lowered in the mutants, in the order R257K > R257Q > R257E, compared to wild type. Our analysis of the binary oscillations in Chl a fluorescence following pretreatment of cells with BQ showed that turnover of the Q(B) site was relatively unaffected in the three mutants. The mutant D1-R257E had the lowest growth rate and steady-state activity and showed the weakest binary oscillations. We conclude that the size and the charge of the amino acid at the position D1-257 play a role in PS II function by modulating the effective redox potential of the Q(B)/Q(B)(-) pair. We discuss an indirect mechanism mediated through electrostatic and/or surface charge effects and the possibility of more pleiotropic effects arising from decreased stability of the D1/D2 and D1/CP47 interfaces.

The Q-cycle reviewed: How well does a monomeric mechanism of the bc(1) complex account for the function of a dimeric complex?

Recent progress in understanding the Q-cycle mechanism of the bc(1) complex is reviewed. The data strongly support a mechanism in which the Q(o)-site operates through a reaction in which the first electron transfer from ubiquinol to the oxidized iron-sulfur protein is the rate-determining step for the overall process. The reaction involves a proton-coupled electron transfer down a hydrogen bond between the ubiquinol and a histidine ligand of the [2Fe-2S] cluster, in which the unfavorable protonic configuration contributes a substantial part of the activation barrier. The reaction is endergonic, and the products are an unstable ubisemiquinone at the Q(o)-site, and the reduced iron-sulfur protein, the extrinsic mobile domain of which is now free to dissociate and move away from the site to deliver an electron to cyt c(1) and liberate the H(+). When oxidation of the semiquinone is prevented, it participates in bypass reactions, including superoxide generation if O(2) is available. When the b-heme chain is available as an acceptor, the semiquinone is oxidized in a process in which the proton is passed to the glutamate of the conserved -PEWY- sequence, and the semiquinone anion passes its electron to heme b(L) to form the product ubiquinone. The rate is rapid compared to the limiting reaction, and would require movement of the semiquinone closer to heme b(L) to enhance the rate constant. The acceptor reactions at the Q(i)-site are still controversial, but likely involve a "two-electron gate" in which a stable semiquinone stores an electron. Possible mechanisms to explain the cyt b(150) phenomenon are discussed, and the information from pulsed-EPR studies about the structure of the intermediate state is reviewed. The mechanism discussed is applicable to a monomeric bc(1) complex. We discuss evidence in the literature that has been interpreted as shown that the dimeric structure participates in a more complicated mechanism involving electron transfer across the dimer interface. We show from myxothiazol titrations and mutational analysis of Tyr-199, which is at the interface between monomers, that no such inter-monomer electron transfer is detected at the level of the b(L) hemes. We show from analysis of strains with mutations at Asn-221 that there are coulombic interactions between the b-hemes in a monomer. The data can also be interpreted as showing similar coulombic interaction across the dimer interface, and we discuss mechanistic implications.

Marcus treatment of endergonic reactions: a commentary.

Two forms of the equation for expression of the rate constant for electron transfer through a Marcus-type treatment are discussed. In the first (exergonic) form, the Arrhenius exponential term was replaced by its classical Marcus term; in the second (endergonic) form, the forward rate constant was replaced by the reverse rate constant (the forward rate constant in the exergonic direction), which was expanded to an equivalent Marcus term and multiplied by the equilibrium constant. When the classical Marcus treatment was used, these two forms of the rate equation give identical curves relating the logarithm of the rate constant to the driving force. The Marcus term for the relation between activation free-energy, DeltaG#, reorganization energy, lambda, and driving force, DeltaG(o), derived from parabolas for the reactant and product states, was identical when starting from exergonic or endergonic parabolas. Moser and colleagues introduced a quantum mechanical correction factor to the Marcus term in order to fit experimental data. When the same correction factor was applied in the treatment for the endergonic direction by Page and colleagues, a different curve was obtained from that found with the exergonic form. We show that the difference resulted from an algebraic error in development of the endergonic equation.

Hydrogen bonds between nitrogen donors and the semiquinone in the Qi-site of the bc1 complex.

The ubisemiquinone stabilized at the Qi-site of the bc1 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 14N and 15N have been studied by X-band (approximately 9.7 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 14N involved in H-bond formation and to assign it unambiguously to the Nepsilon of His-217. This tensor has distinct characteristics in comparison with H-bonds between semiquinones and Ndelta in other quinone-processing sites. The experiments with 15N showed that the Nepsilon 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 Nepsilon 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+/-0.13A. 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.