Quinol Binding Site Research Articles

A 2.2 Å cryoEM structure of a quinol-dependent NO Reductase shows close similarity to respiratory oxidases

Quinol-dependent nitric oxide reductases (qNORs) are considered members of the respiratory heme-copper oxidase superfamily, are unique to bacteria, and are commonly found in pathogenic bacteria where they play a role in combating the host immune response. qNORs are also essential enzymes in the denitrification pathway, catalysing the reduction of nitric oxide to nitrous oxide. Here, we determine a 2.2 Å cryoEM structure of qNOR from Alcaligenes xylosoxidans, an opportunistic pathogen and a denitrifying bacterium of importance in the nitrogen cycle. This high-resolution structure provides insight into electron, substrate, and proton pathways, and provides evidence that the quinol binding site not only contains the conserved His and Asp residues but also possesses a critical Arg (Arg720) observed in cytochrome bo3, a respiratory quinol oxidase.

The cryoEM structure of cytochrome bd from C. glutamicum provides novel insights into structural properties of actinobacterial terminal oxidases.

Cytochromes bd are essential for microaerobic respiration of many prokaryotes including a number of human pathogens. These enzymes catalyze the reduction of molecular oxygen to water using quinols as electron donors. Their importance for prokaryotic survival and the absence of eukaryotic homologs make these enzyme ideal targets for antimicrobial drugs. Here, we determined the cryoEM structure of the menaquinol-oxidizing cytochrome bd-type oxygen reductase of the facultative anaerobic Actinobacterium Corynebacterium glutamicum at a resolution of 2.7Å. The obtained structure adopts the signature pseudosymmetrical heterodimeric architecture of canonical cytochrome bd oxidases formed by the core subunits CydA and CydB. No accessory subunits were identified for this cytochrome bd homolog. The two b-type hemes and the oxygen binding heme d are organized in a triangular geometry with a protein environment around these redox cofactors similar to that of the closely related cytochrome bd from M. tuberculosis. We identified oxygen and a proton conducting channels emerging from the membrane space and the cytoplasm, respectively. Compared to the prototypical enzyme homolog from the E. coli, the most apparent difference is found in the location and size of the proton channel entry site. In canonical cytochrome bd oxidases quinol oxidation occurs at the highly flexible periplasmic Q-loop located in the loop region between TMHs six and seven. An alternative quinol-binding site near heme b 595 was previously identified for cytochrome bd from M. tuberculosis. We discuss the relevance of the two quinol oxidation sites in actinobacterial bd-type oxidases and highlight important differences that may explain functional and electrochemical differences between C. glutamicum and M. tuberculosis. This study expands our current understanding of the structural diversity of actinobacterial and proteobacterial cytochrome bd oxygen reductases and provides deeper insights into the unique structural and functional properties of various cytochrome bd variants from different phylae.

Evolution of quinol oxidation within the heme‑copper oxidoreductase superfamily

The heme‑copper oxidoreductase (HCO) superfamily is a large superfamily of terminal respiratory enzymes that are widely distributed across the three domains of life. The superfamily includes biochemically diverse oxygen reductases and nitric oxide reductases that are pivotal in the pathways of aerobic respiration and denitrification. The adaptation of HCOs to use quinol as the electron donor instead of cytochrome c has significant implication for the respiratory flexibility and energetic efficiency of the respiratory chains that include them. In this work, we explore the adaptation of this scaffold to two different electron donors, cytochromes c and quinols, with extensive sequence analysis of these enzymes from publicly available datasets. Our work shows that quinol oxidation evolved independently within the HCO superfamily at least seven times. Enzymes from only two of these independently evolved clades have been biochemically well-characterized. Combining structural modeling with sequence analysis, we identify putative quinol binding sites in each of the novel quinol oxidases. Our analysis of experimental and modeling data suggests that the quinol binding site appears to have evolved at the same structural position within the scaffold more than once.

Preparations of Terminal Oxidase Cytochrome bd-II Isolated from Escherichia coli Reveal Significant Hydrogen Peroxide Scavenging Activity.

Cytochrome bd-II is one of the three terminal quinol oxidases of the aerobic respiratory chain of Escherichia coli. Preparations of the detergent-solubilized untagged bd-II oxidase isolated from the bacterium were shown to scavenge hydrogen peroxide (H2O2) with high rate producing molecular oxygen (O2). Addition of H2O2 to the same buffer that does not contain enzyme or contains thermally denatured cytochrome bd-II does not lead to any O2 production. The latter observation rules out involvement of adventitious transition metals bound to the protein. The H2O2-induced O2 production is not susceptible to inhibition by N-ethylmaleimide (the sulfhydryl binding compound), antimycin A (the compound that binds specifically to a quinol binding site), and CO (diatomic gas that binds specifically to the reduced heme d). However, O2 formation is inhibited by cyanide (IC50 = 4.5 ± 0.5 µM) and azide. Addition of H2O2 in the presence of dithiothreitol and ubiquinone-1 does not inactivate cytochrome bd-II and apparently does not affect the O2 reductase activity of the enzyme. The ability of cytochrome bd-II to detoxify H2O2 could play a role in bacterial physiology by conferring resistance to the peroxide-mediated stress.

Structure of Mycobacterium tuberculosis cytochrome bcc in complex with Q203 and TB47, two anti-TB drug candidates.

Pathogenic mycobacteria pose a sustained threat to global human health. Recently, cytochrome bcc complexes have gained interest as targets for antibiotic drug development. However, there is currently no structural information for the cytochrome bcc complex from these pathogenic mycobacteria. Here, we report the structures of Mycobacterium tuberculosis cytochrome bcc alone (2.68 Å resolution) and in complex with clinical drug candidates Q203 (2.67 Å resolution) and TB47 (2.93 Å resolution) determined by single-particle cryo-electron microscopy. M. tuberculosis cytochrome bcc forms a dimeric assembly with endogenous menaquinone/menaquinol bound at the quinone/quinol-binding pockets. We observe Q203 and TB47 bound at the quinol-binding site and stabilized by hydrogen bonds with the side chains of QcrBThr313 and QcrBGlu314, residues that are conserved across pathogenic mycobacteria. These high-resolution images provide a basis for the design of new mycobacterial cytochrome bcc inhibitors that could be developed into broad-spectrum drugs to treat mycobacterial infections.

Evolution of the cytochrome bd oxygen reductase superfamily and the function of CydAA' in Archaea.

Cytochrome bd-type oxygen reductases (cytbd) belong to one of three enzyme superfamilies that catalyze oxygen reduction to water. They are widely distributed in Bacteria and Archaea, but the full extent of their biochemical diversity is unknown. Here we used phylogenomics to identify three families and several subfamilies within the cytbd superfamily. The core architecture shared by all members of the superfamily consists of four transmembrane helices that bind two active site hemes, which are responsible for oxygen reduction. While previously characterized cytochrome bd-type oxygen reductases use quinol as an electron donor to reduce oxygen, sequence analysis shows that only one of the identified families has a conserved quinol binding site. The other families are missing this feature, suggesting that they use an alternative electron donor. Multiple gene duplication events were identified within the superfamily, resulting in significant evolutionary and structural diversity. The CydAA' cytbd, found exclusively in Archaea, is formed by the co-association of two superfamily paralogs. We heterologously expressed CydAA' from Caldivirga maquilingensis and demonstrated that it performs oxygen reduction with quinol as an electron donor. Strikingly, CydAA' is the first isoform of cytbd containing only b-type hemes shown to be active when isolated from membranes, demonstrating that oxygen reductase activity in this superfamily is not dependent on heme d.

A ferredoxin bridge connects the two arms of plant mitochondrial complex I.

Mitochondrial complex I is the main site for electron transfer to the respiratory chain and generates much of the proton gradient across the inner mitochondrial membrane. Complex I is composed of two arms, which form a conserved L-shape. We report the structures of the intact, 47-subunit mitochondrial complex I from Arabidopsis thaliana and the 51-subunit complex I from the green alga Polytomella sp., both at around 2.9 Å resolution. In both complexes, a heterotrimeric γ-carbonic anhydrase domain is attached to the membrane arm on the matrix side. Two states are resolved in A. thaliana complex I, with different angles between the two arms and different conformations of the ND1 (NADH dehydrogenase subunit 1) loop near the quinol binding site. The angle appears to depend on a bridge domain, which links the peripheral arm to the membrane arm and includes an unusual ferredoxin. We propose that the bridge domain participates in regulating the activity of plant complex I.

The carboxy-terminal insert in the Q-loop is needed for functionality of Escherichia coli cytochrome bd-I

Cytochrome bd, a component of the prokaryotic respiratory chain, is important under physiological stress and during pathogenicity. Electrons from quinol substrates are passed on via heme groups in the CydA subunit and used to reduce molecular oxygen. Close to the quinol binding site, CydA displays a periplasmic hydrophilic loop called Q-loop that is essential for quinol oxidation. In the carboxy-terminal part of this loop, CydA from Escherichia coli and other proteobacteria harbors an insert of ~60 residues with unknown function. In the current work, we demonstrate that growth of the multiple-deletion strain E. coli MB43∆cydA (∆cydA∆cydB∆appB∆cyoB∆nuoB) can be enhanced by transformation with E. coli cytochrome bd-I and we utilize this system for assessment of Q-loop mutants. Deletion of the cytochrome bd-I Q-loop insert abolished MB43∆cydA growth recovery. Swapping the cytochrome bd-I Q-loop for the Q-loop from Geobacillus thermodenitrificans or Mycobacterium tuberculosis CydA, which lack the insert, did not enhance the growth of MB43∆cydA, whereas swapping for the Q-loop from E. coli cytochrome bd-II recovered growth. Alanine scanning experiments identified the cytochrome bd-I Q-loop insert regions Ile318-Met322, Gln338-Asp342, Tyr353-Leu357, and Thr368-Ile372 as important for enzyme functionality. Those mutants that completely failed to recover growth of MB43∆cydA also lacked oxygen consumption activity and heme absorption peaks. Moreover, we were not able to isolate cytochrome bd-I from these inactive mutants. The results indicate that the cytochrome bd Q-loop exhibits low plasticity and that the Q-loop insert in E. coli is needed for complete, stable, assembly of cytochrome bd-I.

Structure of the cytochrome aa3-600 heme-copper menaquinol oxidase bound to inhibitor HQNO shows TM0 is part of the quinol binding site

Virtually all proton-pumping terminal respiratory oxygen reductases are members of the heme-copper oxidoreductase superfamily. Most of these enzymes use reduced cytochrome c as a source of electrons, but a group of enzymes have evolved to directly oxidize membrane-bound quinols, usually menaquinol or ubiquinol. All of the quinol oxidases have an additional transmembrane helix (TM0) in subunit I that is not present in the related cytochrome c oxidases. The current work reports the 3.6-Å-resolution X-ray structure of the cytochrome aa3 -600 menaquinol oxidase from Bacillus subtilis containing 1 equivalent of menaquinone. The structure shows that TM0 forms part of a cleft to accommodate the menaquinol-7 substrate. Crystals which have been soaked with the quinol-analog inhibitor HQNO (N-oxo-2-heptyl-4-hydroxyquinoline) or 3-iodo-HQNO reveal a single binding site where the inhibitor forms hydrogen bonds to amino acid residues shown previously by spectroscopic methods to interact with the semiquinone state of menaquinone, a catalytic intermediate.

Structural basis for energy transduction by respiratory alternative complex III

Electron transfer in respiratory chains generates the electrochemical potential that serves as energy source for the cell. Prokaryotes can use a wide range of electron donors and acceptors and may have alternative complexes performing the same catalytic reactions as the mitochondrial complexes. This is the case for the alternative complex III (ACIII), a quinol:cytochrome c/HiPIP oxidoreductase. In order to understand the catalytic mechanism of this respiratory enzyme, we determined the structure of ACIII from Rhodothermus marinus at 3.9 Å resolution by single-particle cryo-electron microscopy. ACIII presents a so-far unique structure, for which we establish the arrangement of the cofactors (four iron–sulfur clusters and six c-type hemes) and propose the location of the quinol-binding site and the presence of two putative proton pathways in the membrane. Altogether, this structure provides insights into a mechanism for energy transduction and introduces ACIII as a redox-driven proton pump.

Three-dimensional models of Mycobacterium tuberculosis proteins Rv1555, Rv1554 and their docking analyses with sildenafil, tadalafil, vardenafil drugs, suggest interference with quinol binding likely to affect protein\u2019s function

BackgroundEarlier based on bioinformatics analyses, we had predicted the Mycobacterium tuberculosis (M.tb) proteins; Rv1555 and Rv1554, among the potential new tuberculosis drug targets. According to the ‘TB-drugome’ the Rv1555 protein is ‘druggable’ with sildenafil (Viagra), tadalafil (Cialis) and vardenafil (Levitra) drugs. In the present work, we intended to understand via computer modeling studies, how the above drugs are likely to inhibit the M.tb protein’s function.ResultsThe three-dimensional computer models for M.tb proteins; Rv1555 and Rv1554 constructed on the template of equivalent membrane anchor subunits of the homologous E.coli quinol fumarate reductase respiratory protein complex, followed by drug docking analyses, suggested that the binding of above drugs interferes with quinol binding sites. Also, we experimentally observed the in-vitro growth inhibition of E.coli bacteria containing the homologous M.tb protein sequences with sildenafil and tadalafil drugs.ConclusionsThe predicted binding sites of the drugs is likely to affect the above M.tb proteins function as quinol binding is known to be essential for electron transfer function during anaerobic respiration in the homologous E.coli protein complex. Therefore, sildenafil and related drugs currently used in the treatment of male erectile dysfunction targeting the human phosphodiesterase 5 enzyme may be evaluated for their plausible role as repurposed drugs to treat human tuberculosis.

Structural mapping of semiquinone catalytic intermediates at the quinol binding site of the nitrate reductase A from Escherichia coli by selective 15N labeling and pulsed EPR

Respiratory supercomplexes have been extensively studied in mitochondria, but much less concerned in prokaryotes. Here we present the detailed characterization work on a supercomplex III/IV isolated from native membranes of eubacterium Aquifex aeolicus. This supercomplex III/IV survives fromdetergent solubilization and ismonodispersed in both ion-exchange and size-exclusion chromatography. It consists of cytochrome b, cytochrome c1 and Rieske protein from the cytochrome bc1 complex and subunits I, II and IIa from the cytochrome oxidase. Major subunits are clearly visible on SDS-PAGE and later on identified by MALDI-MS. Activity assay showed that this supercomplex has higher turnover numbers than the purified cytochrome oxidase, which was called “Cox2” in our former work [1]. The stoichiometry of the supercomplex has been characterized as a homo-dimeric bc1 complex and a monomeric oxidase based on heme ratios, protein amounts and metal contents. Functional analysis on the supercomplex III2IV indicates potential interaction between the cytochrome bc1 complex and the cytochrome oxidase. In order to investigate the potential interaction, crosslinking work of the supercomplex has been performed, antibodies against protein subunits have been produced, and mass spectrometry has been applied. Further functional analysis of the supercomplex would be done as soon as possible aiming to get a picture of the supercomplex functional organization.

A functional description of CymA, an electron-transfer hub supporting anaerobic respiratory flexibility in Shewanella

CymA (tetrahaem cytochrome c) is a member of the NapC/NirT family of quinol dehydrogenases. Essential for the anaerobic respiratory flexibility of shewanellae, CymA transfers electrons from menaquinol to various dedicated systems for the reduction of terminal electron acceptors including fumarate and insoluble minerals of Fe(III). Spectroscopic characterization of CymA from Shewanella oneidensis strain MR-1 identifies three low-spin His/His co-ordinated c-haems and a single high-spin c-haem with His/H(2)O co-ordination lying adjacent to the quinol-binding site. At pH 7, binding of the menaquinol analogue, 2-heptyl-4-hydroxyquinoline-N-oxide, does not alter the mid-point potentials of the high-spin (approximately -240 mV) and low-spin (approximately -110, -190 and -265 mV) haems that appear biased to transfer electrons from the high- to low-spin centres following quinol oxidation. CymA is reduced with menadiol (E(m) = -80 mV) in the presence of NADH (E(m) = -320 mV) and an NADH-menadione (2-methyl-1,4-naphthoquinone) oxidoreductase, but not by menadiol alone. In cytoplasmic membranes reduction of CymA may then require the thermodynamic driving force from NADH, formate or H2 oxidation as the redox poise of the menaquinol pool in isolation is insufficient. Spectroscopic studies suggest that CymA requires a non-haem co-factor for quinol oxidation and that the reduced enzyme forms a 1:1 complex with its redox partner Fcc3 (flavocytochrome c3 fumarate reductase). The implications for CymA supporting the respiratory flexibility of shewanellae are discussed.

Direct Evidence for Nitrogen Ligation to the High Stability Semiquinone Intermediate in Escherichia coli Nitrate Reductase A

The membrane-bound heterotrimeric nitrate reductase A (NarGHI) catalyzes the oxidation of quinols in the cytoplasmic membrane of Escherichia coli and reduces nitrate to nitrite in the cytoplasm. The enzyme strongly stabilizes a menasemiquinone intermediate at a quinol oxidation site (Q(D)) located in the vicinity of the distal heme b(D). Here molecular details of the interaction between the semiquinone radical and the protein environment have been provided using advanced multifrequency pulsed EPR methods. (14)N and (15)N ESEEM and HYSCORE measurements carried out at X-band ( approximately 9.7 GHz) on the wild-type enzyme or the enzyme uniformly labeled with (15)N nuclei reveal an interaction between the semiquinone and a single nitrogen nucleus. The isotropic hyperfine coupling constant A(iso)((14)N) approximately 0.8 MHz shows that it occurs via an H-bond to one of the quinone carbonyl group. Using (14)N ESEEM and HYSCORE spectroscopies at a lower frequency (S-band, approximately 3.4 GHz), the (14)N nuclear quadrupolar parameters of the interacting nitrogen nucleus (kappa = 0.49, eta = 0.50) were determined and correspond to those of a histidine N(delta), assigned to the heme b(D) ligand His-66 residue. Moreover S-band (15)N ESEEM spectra enabled us to directly measure the anisotropic part of the nitrogen hyperfine interaction (T((15)N) = 0.16 MHz). A distance of approximately 2.2 Abetween the carbonyl oxygen and the nitrogen could then be calculated. Mechanistic implications of these results are discussed in the context of the peculiar properties of the menasemiquinone intermediate stabilized at the Q(D) site of NarGHI.

Quinol Oxidation by c-Type Cytochromes: Structural Characterization of the Menaquinol Binding Site of NrfHA

Membrane-bound cytochrome c quinol dehydrogenases play a crucial role in bacterial respiration by oxidizing menaquinol and transferring electrons to various periplasmic oxidoreductases. In this work, the menaquinol oxidation site of NrfH was characterized by the determination of the X-ray structure of Desulfovibrio vulgaris NrfHA nitrite reductase complex bound to 2-heptyl-4-hydroxyquinoline-N-oxide, which is shown to act as a competitive inhibitor of NrfH quinol oxidation activity. The structure, at 2.8-Å resolution, reveals that the inhibitor binds close to NrfH heme 1, where it establishes polar contacts with two essential residues: Asp89, the residue occupying the heme distal ligand position, and Lys82, a strictly conserved residue. The menaquinol binding cavity is largely polar and has a wide opening to the protein surface. Coarse-grained molecular dynamics simulations suggest that the quinol binding site of NrfH and several other respiratory enzymes lie in the head group region of the membrane, which probably facilitates proton transfer to the periplasm. Although NrfH is not a multi-span membrane protein, its quinol binding site has several characteristics similar to those of quinone binding sites previously described. The data presented here provide the first characterization of the quinol binding site of the cytochrome c quinol dehydrogenase family.

S- and N-Oxide Reductases.

Escherichia coli is a versatile facultative anaerobe that can respire on a number of terminal electron acceptors, including oxygen, fumarate, nitrate, and S- and N-oxides. Anaerobic respiration using S- and N-oxides is accomplished by enzymatic reduction of these substrates by dimethyl sulfoxide reductase (DmsABC) and trimethylamine N-oxide reductase (TorCA). Both DmsABC and TorCA are membrane-associated redox enzymes that couple the oxidation of menaquinol to the reduction of S- and N-oxides in the periplasm. DmsABC is membrane bound and is composed of a membrane-extrinsic dimer with a 90.4-kDa catalytic subunit (DmsA) and a 23.1-kDa electron transfer subunit (DmsB). These subunits face the periplasm and are held to the membrane by a 30.8-kDa membrane anchor subunit (DmsC). The enzyme provides the scaffold for an electron transfer relay composed of a quinol binding site, five [4Fe-4S] clusters, and a molybdo-bis(molybdopterin guanine dinucleotide) (present nomenclature: Mo-bis-pyranopterin) (Mo-bisMGD) cofactor. TorCA is composed of a soluble periplasmic subunit (TorA, 92.5 kDa) containing a Mo-bis-MGD. TorA is coupled to the quinone pool via a pentaheme c subunit (TorC, 40.4 kDa) in the membrane. Both DmsABC and TorCA require system-specific chaperones (DmsD or TorD) for assembly, cofactor insertion, and/or targeting to the Tat translocon. In this chapter, we discuss the complex regulation of the dmsABC and torCAD operons, the poorly understood paralogues, and what is known about the assembly and translocation to the periplasmic space by the Tat translocon.

High-Stability Semiquinone Intermediate in Nitrate Reductase A (NarGHI) from Escherichia coli Is Located in a Quinol Oxidation Site Close to Heme bD

Quinol/nitrate oxidoreductase (NarGHI) is the first enzyme involved in respiratory denitrification in prokaryotes. Although this complex in E. coli is known to operate with both ubi and menaquinones, the location and the number of quinol binding sites remain elusive. NarGHI strongly stabilizes a semiquinone radical located within the dihemic anchor subunit NarI. To identify its location and function, we used a combination of mutagenesis, kinetics, EPR, and ENDOR spectroscopies. For the NarGHIH66Y and NarGHIH187Y mutants lacking the distal heme bD, no EPR signal of the semiquinone was observed. In contrast, a semiquinone was detected in the NarGHIH56Y mutant lacking the proximal heme bP. Its thermodynamic properties and spectroscopic characteristics, as revealed by Q-band EPR and ENDOR spectroscopies, are identical to those observed in the native enzyme. The substitution by Ala of the Lys86 residue close to heme bD, which was previously proposed to be in a quinol oxidation site of NarGHI (QD), also leads to the loss of the EPR signal of the semiquinone, although both hemes are present. Enzymatic assays carried out on the NarGHIK86A mutant reveal that the substitution dramatically reduces the rate of oxidation of both mena and ubiquinol analogues. These observations demonstrate that the semiquinone observed in NarI is strongly associated with heme bD and that Lys86 is required for its stabilization. Overall, our results indicate that the semiquinone is located within the quinol oxidation site QD. Details of the possible binding motif of the semiquinone and mechanistic implications are discussed.

Glutamate 107 in Subunit I of the Cytochrome bd Quinol Oxidase from Escherichia coli Is Protonated and near the Heme d/Heme b595 Binuclear Center

Cytochrome bd is a quinol oxidase from Escherichia coli, which is optimally expressed under microaerophilic growth conditions. The enzyme catalyzes the two-electron oxidation of either ubiquinol or menaquinol in the membrane and scavenges O2 at low concentrations, reducing it to water. Previous work has shown that, although cytochrome bd does not pump protons, turnover is coupled to the generation of a proton motive force. The generation of a proton electrochemical gradient results from the release of protons from the oxidation of quinol to the periplasm and the uptake of protons used to form H2O from the cytoplasm. Because the active site has been shown to be located near the periplasmic side of the membrane, a proton channel must facilitate the delivery of protons from the cytoplasm to the site of water formation. Two conserved glutamic acid residues, E107 and E99, are located in transmembrane helix III in subunit I and have been proposed to form part of this putative proton channel. In the current work, it is shown that mutations in either of these residues results in the loss of quinol oxidase activity and can result in the loss of the two hemes at the active site, hemes d and b595. One mutant, E107Q, while being totally inactive, retains the hemes. Fourier transform infrared (FTIR) redox difference spectroscopy has identified absorption bands from the COOH group of E107. The data show that E107 is protonated at pH 7.6 and that it is perturbed by the reduction of the heme d/heme b595 binuclear center at the active site. In contrast, mutation of an acidic residue known to be at or near the quinol-binding site (E257A) also inactivates the enzyme but has no substantial influence on the FTIR redox difference spectrum. Mutagenesis shows that there are several acidic residues, including E99 and E107 as well as D29 (in CydB), which are important for the assembly or stability of the heme d/heme b595 active site.

Probing the Ubiquinol-Binding Site in Cytochromebdby Site-Directed Mutagenesis

To probe the structure of the quinol oxidation site in loop VI/VII of the Escherichia coli cytochrome bd, we substituted three conserved residues (Gln249, Lys252, and Glu257) in the N-terminal region and three glutamates (Glu278, Glu279, and Glu280) in the first internal repeat. We found that substitutions of Glu257 by Ala or Gln, and Glu279 and Glu280 by Gln, severely reduced the oxidase activity and the expression level of cytochrome bd. In contrast, Lys252 mutations reduced only the oxidase activity. Blue shifts in the 440 and 630 nm peaks of the reduced Lys252 mutants and in the 561 nm peak of the reduced Glu257 mutants indicate the proximity of Lys252 to the heme b(595)-d binuclear center and Glu257 to heme b(558), respectively. Perturbations of reduced heme b(558) upon binding of aurachin D support structural changes in the quinol-binding site of the mutants. Substitutions of Lys252 and Glu257 caused large changes in kinetic parameters for the ubiquinol-1 oxidation. These results indicate that Lys252 and Glu257 in the N-terminal region of the Q-loop are involved in the quinol oxidation by bd-type terminal oxidase.

Mass Spectrometric Analysis of the Ubiquinol-binding Site in Cytochrome bd from Escherichia coli

Cytochrome bd is a heterodimeric terminal ubiquinol oxidase in the aerobic respiratory chain of Escherichia coli. For understanding the unique catalytic mechanism of the quinol oxidation, mass spectrometry was used to identify amino acid residue(s) that can be labeled with a reduced form of 2-azido-3-methoxy-5-methyl-6-geranyl-1,4-benzoquinone or 2-methoxy-3-azido-5-methyl-6-geranyl-1,4-benzoquinone. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry demonstrated that the photo inactivation of ubiquinol-1 oxidase activity was accompanied by the labeling of subunit I with both azidoquinols. The cross-linked domain was identified by reverse-phase high performance liquid chromatography of subunit I peptides produced by in-gel double digestion with lysyl endopeptidase and endoproteinase Asp-N. Electrospray ionization quadrupole time-of-flight mass spectrometry determined the amino acid sequence of the peptide (m/z 1047.5) to be Glu(278)-Lys(283), where a photoproduct of azido-Q(2) was linked to the carboxylic side chain of I-Glu(280). This study demonstrated directly that the N-terminal region of periplasmic loop VI/VII (Q-loop) is a part of the quinol oxidation site and indicates that the 2- and 3-methoxy groups of the quinone ring are in the close vicinity of I-Glu(280).