B-type Heme Research Articles

Direct Electron Transfer of Cellobiose Dehydrogenase on Positively Charged Polyethyleneimine Gold Nanoparticles.

Efficient conjugation between biomolecules and electrode materials is one of the main challenges in the field of biosensors. Cellobiose dehydrogenase (CDH) is a monomeric enzyme, which consists of two separate domains: one catalytic dehydrogenase domain (DHCDH ) carrying strongly bound flavin adenine dinucleotide (FAD) in the active site and a cytochrome domain (CYTCDH ) carrying a b-type heme connected by a flexible linker region. Herein, we report on the development of a lactose biosensor, based on direct electron transfer (DET) from CDH from Phanerochaete sordida (PsCDH) electrostatically attached onto polyethyleneimine-stabilized gold nanoparticles (PEI@AuNPs) used to cover a conventional polycrystalline solid gold disk electrode. PEI@AuNPs were synthesized in aqueous solution using PEI as reducing agent for AuIII and as stabilizer for the nanoparticles. The heterogeneous electron-transfer (ET) rate (ks ) for the redox reaction of immobilized PsCDH at the modified electrodes was calculated based on the Laviron theory and was found to be (39.6±2.5) s-1 . The proposed lactose biosensor exhibits good long term stability as well as high and reproducible sensitivity to lactose with a response time less than 5 s and a linear range from 1 to 100 μm.

Biochemical and spectroscopic characterization of purified Latex Clearing Protein (Lcp) from newly isolated rubber degrading Rhodococcus rhodochrous strain RPK1 reveals novel properties of Lcp.

BackgroundBiodegradation of rubber (polyisoprene) is initiated by oxidative cleavage of the polyisoprene backbone and is performed either by an extracellular rubber oxygenase (RoxA) from Gram-negative rubber degrading bacteria or by a latex clearing protein (Lcp) secreted by Gram-positive rubber degrading bacteria. Only little is known on the biochemistry of polyisoprene cleavage by Lcp and on the types and functions of the involved cofactors.ResultsA rubber-degrading bacterium was isolated from the effluent of a rubber-processing factory and was taxonomically identified as a Rhodococcus rhodochrous species. A gene of R. rhodochrous RPK1 that coded for a polyisoprene-cleaving latex clearing protein (lcpRr) was identified, cloned, expressed in Escherichia coli and purified. Purified LcpRr had a specific activity of 3.1 U/mg at 30 °C and degraded poly(1,4-cis-isoprene) to a mixture of oligoisoprene molecules with terminal keto and aldehyde groups. The pH optimum of LcpRr was higher (pH 8) than for other rubber-cleaving enzymes (≈ pH 7). UVvis spectroscopic analysis of LcpRr revealed a cytochrome-specific absorption spectrum with an additional feature at long wavelengths that has not been observed for any other rubber-cleaving enzyme. The presence of one b-type haem in LcpRr as a co-factor was confirmed by (i) metal analysis, (ii) solvent extraction, (iii) bipyridyl assay and (iv) detection of haem-b specific m/z values via mass-spectrometry.ConclusionsOur data point to substantial differences in the active sites of Lcp proteins obtained from different rubber degrading bacteria.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-016-0703-x) contains supplementary material, which is available to authorized users.

Characterization of a Single b-type Heme, FAD, and Metal Binding Sites in the Transmembrane Domain of Six-transmembrane Epithelial Antigen of the Prostate (STEAP) Family Proteins

Six-transmembrane epithelial antigen of the prostate 3 (Steap3) is the major ferric reductase in developing erythrocytes. Steap family proteins are defined by a shared transmembrane domain that in Steap3 has been shown to function as a transmembrane electron shuttle, moving cytoplasmic electrons derived from NADPH across the lipid bilayer to the extracellular face where they are used to reduce Fe(3+) to Fe(2+) and potentially Cu(2+) to Cu(1+). Although the cytoplasmic N-terminal oxidoreductase domain of Steap3 and Steap4 are relatively well characterized, little work has been done to characterize the transmembrane domain of any member of the Steap family. Here we identify high affinity FAD and iron biding sites and characterize a single b-type heme binding site in the Steap3 transmembrane domain. Furthermore, we show that Steap3 is functional as a homodimer and that it utilizes an intrasubunit electron transfer pathway through the single heme moiety rather than an intersubunit electron pathway through a potential domain-swapped dimer. Importantly, the sequence motifs in the transmembrane domain that are associated with the FAD and metal binding sites are not only present in Steap2 and Steap4 but also in Steap1, which lacks the N-terminal oxidoreductase domain. This strongly suggests that Steap1 harbors latent oxidoreductase activity.

The homopentameric chlorite dismutase from Magnetospirillum sp.

Chlorite dismutase (Cld) is a b-type heme containing enzyme that catalyzes the reduction of chlorite into chloride plus dioxygen. This enzyme has gained attention because it can be used in the development of bioremediation processes, biosensors, and controlled dioxygen production. In the present work, Cld was purified from Magnetospirillum sp. cells cultured anaerobically with acetate/perchlorate until stationary phase. Biochemical, spectroscopic and X-ray crystallography methods showed that Cld from Magnetospirillum sp. is a ~140kDa homopentamer comprising ~27.8kDa monomers. Preliminary X-ray crystallography studies confirmed the quaternary structure and the presence of one b-type heme per monomer. The EPR spectroscopic signature of the as-purified Cld samples is affected by the buffer composition used during the purification. Potassium phosphate buffer is the only buffer that affected neither the spectral nor the kinetic properties of Cld. Kinetic studies in solution revealed that Cld from Magnetospirillum sp. decomposes chlorite at high turnover rates with optimal pH6.0. A temperature below 10°C is required to avoid enzyme inactivation due to cofactor bleaching during turnover, and to achieve full substrate consumption. Cld kinetic parameters were not affected when kinetic assays were performed in the presence of air or under argon atmosphere, but chloride is a weak mixed inhibitor that modifies the EPR signal of as-prepared samples.

Ligand-Binding and Substrate Turnover of a Heme Peroxidase from the Diatom Thalassiosira Pseudonana

Iron is sparingly soluble in water; therefore, iron-containing enzymes in marine organisms such as diatoms must have critical physiological roles to justify the use of such a precious resource. We have identified a putative heme peroxidase of unknown function, Tp 21683, from the published genome of the model diatom Thalassiosira pseudonana. Conserved domain analysis suggests Tp 21683 consists of an N-terminal kringle domain, likely involved in ligand binding and localization, and a C-terminal b-type heme peroxidase domain. To dissect the function of the two domains of Tp 21683, we have optimized recombinant expression in Escherichia coli and subsequent refolding and purification of the isolated kringle and peroxidase domains. We report preliminary studies to identify the ligand-binding and peroxidase turnover properties of Tp 21683 toward a better understanding of the physiological role of peroxidases in diatoms. We show by intrinsic tryptophan fluorescence that unlike canonical kringle domains from human plasminogen, the kringle domain of Tp 21683 does not show measurable binding to lysine. However, the kringle domain appears to bind to the oleic acid with a Kd of approximately 1 μM. The peroxidase domain is able to turn over hydrogen peroxide using both the nonphysiological electron donor ABTS and the aromatic compound guaiacol with kcat of approximately 40 min-1 and 4 min-1, respectively. These data are the first analysis of a recombinant peroxidase from a diatom, and provide a basis for further investigation of the range of possible ligands and chemical reactions catalyzed by Tp 21683.

Thermal Stability of a B-Type Heme Peroxidase from the Psychrophilic Diatom Fragilariopsis Cylindrus

The eukaryotic, unicellular diatom Fragilariopsis cylindrus thrives in marine environments at temperatures near the freezing point of water. As a result, critical enzymes of F. cylindrus must have specifically evolved to maintain flexibility and activity at very low temperatures. Like all photosynthetic organisms, F. cylindrus must also have robust pathways, possibly including peroxidases, to protect its cellular components against oxidative damage. In this study, we report the recombinant expression and purification of a b-type heme peroxidase, Fc 209911, identified from the published genome of F. cylindrus. The enzyme is expressed in the apo form in Escherichia coli, and forms an active holo-enzyme upon reconstitution with hemin. The purified enzyme turns over hydrogen peroxide in the presence of ABTS with modest turnover rates (20 min-1), yet binds hydrogen peroxide with a very high affinity (Km < 100 nM). As predicted, mutation of the conserved histidine H73, which presumably forms a hydrogen bond donor/acceptor in the proximal heme pocket, to alanine decreases hydrogen peroxide binding affinity (∼ 10,000x increase in Km), suggesting that the enzyme has a typical peroxidase fold and mechanism. Thermal unfolding monitored by the intensity of the Soret absorbance band shows a single unfolding transition around 295 K. Analysis of the rate of hydrogen peroxide turnover at a range of temperatures shows maximal activity around 303 K, with a steep decrease in activity as temperature is raised further. The temperature dependence of folding and peroxidase activity confirms that Fc 209911 is optimized to function at low temperatures. Experiments are currently underway to characterize the van’t Hoff enthalpy of the unfolding transition. Future experiments will investigate the basis of the thermal instability of Fc 209911 by designing site-directed mutants to increase the melting temperature.

The trans-membrane cytochrome b561 proteins: structural information and biological function.

Cytochrome b561 (CYB561) proteins are ascorbate reducible, trans-membrane proteins consisting of 200-300 amino acids, about half of which are hydrophobic. The first identified CYB561 protein was discovered more than 40 years ago, and is localized in the chromaffin granule membrane of the mammalian adrenal glands. Proteins with similar structural elements and biophysical and biochemical properties were identified in a wide range of animal and plant phyla in the past 15 years. CYB561 proteins have six trans-membrane helices and two b-type hemes, one on each side of the membrane. The two heme-b centers are coordinated by two pairs of His residues localized in the central four trans-membrane domains, probably very close to the membrane interface. The midpoint redox potentials of the two hemes are above 0 mV and about 100 mV apart from each other. These proteins are different in many respects from the well-known two heme-bcontaining, trans-membrane b-type cytochromes localized in the inner membrane of mitochondria, in the chloroplast thylakoids or in the cell membrane. The atomic-level structure of only one CYB561 protein is available to date. In this paper we discuss in detail the biophysical and biochemical properties of the CYB561 proteins and provide a short overview of their known or putative biological functions and significance.

Q-Site Occupancy Defines Heme Heterogeneity in Escherichia coli Nitrate Reductase A (NarGHI)

The membrane subunit (NarI) of Escherichia coli nitrate reductase A (NarGHI) contains two b-type hemes, both of which are the highly anisotropic low-spin type. Heme bD is distal to NarGH and constitutes part of the quinone binding and oxidation site (Q-site) through the axially coordinating histidine-66 residue and one of the heme bD propionate groups. Bound quinone participates in hydrogen bonds with both the imidazole of His66 and the heme propionate, rendering the EPR spectrum of the heme bD sensitive to Q-site occupancy. As such, we hypothesize that the heterogeneity in the heme bD EPR signal arises from the differential occupancy of the Q-site. In agreement with this, the heterogeneity is dependent upon growth conditions but is still apparent when NarGHI is expressed in a strain lacking cardiolipin. Furthermore, this heterogeneity is sensitive to Q-site variants, NarI-G65A and NarI-K86A, and is collapsible by the binding of inhibitors. We found that the two main gz components of heme bD exhibit differences in reduction potential and pH dependence, which we posit is due to differential Q-site occupancy. Specifically, in a quinone-bound state, heme bD exhibits an Em,8 of -35 mV and a pH dependence of -40 mV pH(-1). In the quinone-free state, however, heme bD titrates with an Em,8 of +25 mV and a pH dependence of -59 mV pH(-1). We hypothesize that quinone binding modulates the electrochemical properties of heme bD as well as its EPR properties.

Direct meso‐Alkynylation of Metalloporphyrins Through Gold Catalysis for Hemoprotein Engineering

A method was developed for the direct functionalization of metalloporphyrins at the methine protons (meso positions) to yield asymmetric alkynylated derivatives by using gold catalysis and hypervalent iodine reagents. This single-step procedure was applied to b-type heme and the product was incorporated into a gas-sensor heme protein. The terminal alkyne allows fluorophore labeling through copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC). Hemoproteins with this type of engineered cofactor have several potential applications in labeling and imaging technologies. Additionally, the alkyne provides a handle for modulating porphyrin electron density, which affects cofactor redox potential and ligand affinity. This method will be helpful for investigating the chemistry of natural heme proteins and for designing artificial variants with altered properties and reactivities.

Ferric human neuroglobin scavenges superoxide to form oxy adduct.

Neuroglobin (Ngb) is the third member of the vertebrate globin family, and the structure was solved as a typical globin fold with a b-type heme. Although it has been proposed that Ngb could be involved in neuroprotection against oxidative stress, the protective mechanism has not been fully identified yet. In order to clarify functions under hypoxic condition, in this study, we focused on the scavenger activity of human Ngb (hNgb) against superoxide. The activity of hNgb for superoxide was evaluated to be 7.4 µM for IC50, the half maximal inhibitory concentration. The result indicates that hNgb can be an anti-oxidant, and the value was almost the same as that of ascorbic acid. In addition, we characterized oxidation states of a heme iron in superoxide-treated hNgb with spectroscopic measurements. Superoxide-treated hNgb in the ferric form was readily converted to the oxygenated ferrous form, and the result suggested that ferric hNgb could scavenge superoxide by change of an oxidation state in a heme iron. Moreover, mutational experiments were performed, and the each variant mutated at 46 and 55 positions suggested a disulfide bond between Cys46 and Cys55 could be essential to be sensors for oxidative stress with the direct binding of superoxide. As a consequence, we concluded that redox changes of the heme iron and the disulfide bond could regulate neuroprotective functions of hNgb, and it suggests that hNgb can afford protection against hypoxic and ischemic stress in the brain.

Dielectric Heterogeneity in the Cytochrome B6F Complex

Electron transfer in the dimeric cytochrome b6f complex, which includes four b-type hemes organized as two pairs in symmetric monomers, was studied by simultaneous measurement of the kinetics of heme reduction by dithionite and an associated amplitude increase of Soret band split circular dichroism (CD) spectra diagnostic of heme-heme exciton interactions, for which similar kinetics were determined. Based on inter-heme distances and orientations from crystal structures of the complex, the increase in the split CD signal is dominated by interaction between the two intra-monomer b-hemes, located on the electrochemically negative and positive sides of the complex, whose midpoint oxidation-reduction potentials, Em, determined by titrations of isolated complex, differ by 75-100 mV. Kinetics are fit best by preferential reduction of the intra-monomer heme pair. Equilibration of transferred electrons would, however, predict preferential reduction of the two higher potential hemes, one in each monomer. Heterogeneity of the dielectric constant is implied, a consequence of structure inhomogeneity, and/or dielectric reorganization in response to electron transfer. The largest dielectric constant exists between the intra-monomer b-hemes, resulting in a lower energy state of the reduced intra-monomer heme pair relative to any other heme pair.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Solution Structure of the PAS Domain of a Thermophilic YybT Protein Homolog Reveals a Potential Ligand-binding Site

The Bacillus subtilis protein YybT (or GdpP) and its homologs were recently established as stress signaling proteins that exert their biological effect by degrading the bacterial messenger cyclic di-AMP. YybT homologs contain a small Per-ARNT-Sim (PAS) domain (~80 amino acids) that can bind b-type heme with 1:1 stoichiometry despite the small size of the domain and the lack of a conserved heme iron-coordinating residue. We determined the solution structure of the PAS domain of GtYybT from Geobacillus thermodenitrificans by NMR spectroscopy to further probe its function. The solution structure confirms that PASGtYybT adopts the characteristic PAS fold composed of a five-stranded antiparallel β sheet and a few short α-helices. One α-helix and three central β-strands of PASGtYybT are noticeably shorter than those of the typical PAS domains. Despite the small size of the protein domain, a hydrophobic pocket is formed by the side chains of nonpolar residues stemming from the β-strands and α-helices. A set of residues in the vicinity of the pocket and in the C-terminal region at the dimeric interface exhibits perturbed NMR parameters in the presence of heme or zinc protoporphyrin. Together, the results unveil a compact PAS domain with a potential ligand-binding pocket and reinforce the view that the PASYybT domains function as regulatory domains in the modulation of cellular cyclic di-AMP concentration.

The Heme Chaperone ApoCcmE Forms a Ternary Complex with CcmI and Apocytochrome c

Cytochrome c maturation (Ccm) is a post-translational process that occurs after translocation of apocytochromes c to the positive (p) side of energy-transducing membranes. Ccm is responsible for the formation of covalent bonds between the thiol groups of two cysteines residues at the heme-binding sites of the apocytochromes and the vinyl groups of heme b (protoporphyrin IX-Fe). Among the proteins (CcmABCDEFGHI and CcdA) required for this process, CcmABCD are involved in loading heme b to apoCcmE. The holoCcmE thus formed provides heme b to the apocytochromes. Catalysis of the thioether bonds between the apocytochromes c and heme b is mediated by the heme ligation core complex, which in Rhodobacter capsulatus contains at least the CcmF, CcmH, and CcmI components. In this work we show that the heme chaperone apoCcmE binds to the apocytochrome c and the apocytochrome c chaperone CcmI to yield stable binary and ternary complexes in the absence of heme in vitro. We found that during these protein-protein interactions, apoCcmE favors the presence of a disulfide bond at the apocytochrome c heme-binding site. We also establish using detergent-dispersed membranes that apoCcmE interacts directly with CcmI and CcmH of the heme ligation core complex CcmFHI. Implications of these findings are discussed with respect to heme transfer from CcmE to the apocytochromes c during heme ligation assisted by the core complex CcmFHI.

Structure, recombinant expression and mutagenesis studies of the catalase with oxidase activity fromScytalidium thermophilum

Scytalidium thermophilum produces a catalase with phenol oxidase activity (CATPO) that catalyses the decomposition of hydrogen peroxide into oxygen and water and also oxidizes various phenolic compounds. A codon-optimized catpo gene was cloned and expressed in Escherichia coli. The crystal structures of native and recombinant S. thermophilum CATPO and two variants, H82N and V123F, were determined at resolutions of 2.7, 1.4, 1.5 and 1.9 Å, respectively. The structure of CATPO reveals a homotetramer with 698 residues per subunit and with strong structural similarity to Penicillium vitale catalase. The haem component is cis-hydroxychlorin γ-spirolactone, which is rotated 180° with respect to small-subunit catalases. The haem-binding pocket contains two highly conserved water molecules on the distal side. The H82N mutation resulted in conversion of the native d-type haem to a b-type haem. Kinetic studies of the H82N and V123F mutants indicate that both activities are likely to be associated with the haem centre and suggest that the secondary oxidase activity may be a general feature of catalases in the absence of hydrogen peroxide.

Functional Importance of a Pair of Conserved Glutamic Acid Residues and of Ca2+ Binding in the cbb3-Type Oxygen Reductases from Rhodobacter sphaeroides and Vibrio cholerae

The cbb(3)-type cytochrome c oxidases are members of the family of heme-copper proton pumping respiratory oxygen reductases. The structure of the cbb(3)-type oxidase from Pseudomonas stutzeri reveals that, in addition to the six redox-active metal centers (two b-type hemes, three c-type hemes, and Cu(B)), the enzyme also contains at least one Ca(2+). The calcium bridges two propionate carboxyls at the interface between the low-spin heme b and the active-site heme b(3) and, in addition, is ligated to a serine in subunit CcoO and by a glutamate in subunit CcoN. The glutamate that is ligated to Ca(2+) is one of a pair of glutamic acid residues that has previously been suggested to be part of a proton exit pathway for pumped protons. In this work, mutations of these glutamates are investigated in the cbb(3)-type oxidases from Vibrio cholerae and Rhodobacter sphaeroides. Metal analysis shows that each of these wild-type enzymes contains Ca(2+). Mutations of the glutamate expected to ligate the Ca(2+) in each of these enzymes (E126 in V. cholerae and E180 in R. sphaeroides) result in a loss of activity as well as a loss of Ca(2+). Mutations of the nearby glutamate (E129 in V. cholerae and E183 in R. sphaeroides) also resulted in a loss of oxidase activity and a loss of Ca(2+). It is concluded that the Ca(2+) is essential for assembly of the fully functional enzyme and that neither of the glutamates is likely to be part of a pathway for pumped protons within the cbb(3)-type oxygen reductases. A more likely role for these glutamates is the maintenance of the structural integrity of the active conformation of the enzyme.

Expression of Saccharomyces cerevisiae Sdh3p and Sdh4p Paralogs Results in Catalytically Active Succinate Dehydrogenase Isoenzymes

Succinate dehydrogenase (SDH), also known as complex II, is required for respiratory growth; it couples the oxidation of succinate to the reduction of ubiquinone. The enzyme is composed of two domains. A membrane-extrinsic catalytic domain composed of the Sdh1p and Sdh2p subunits harbors the flavin and iron-sulfur cluster cofactors. A membrane-intrinsic domain composed of the Sdh3p and Sdh4p subunits interacts with ubiquinone and may coordinate a b-type heme. In many organisms, including Saccharomyces cerevisiae, possible alternative SDH subunits have been identified in the genome. S. cerevisiae contains one paralog of the Sdh3p subunit, Shh3p (YMR118c), and two paralogs of the Sdh4p subunit, Shh4p (YLR164w) and Tim18p (YOR297c). We cloned and expressed these alternative subunits. Shh3p and Shh4p were able to complement Δsdh3 and Δsdh4 deletion mutants, respectively, and support respiratory growth. Tim18p was unable to do so. Microarray and proteomics data indicate that the paralogs are expressed under respiratory and other more restrictive growth conditions. Strains expressing hybrid SDH enzymes have distinct metabolic profiles that we distinguished by (1)H NMR analysis of metabolites. Surprisingly, the Sdh3p subunit can form SDH isoenzymes with Sdh4p or with Shh4p as well as be a subunit of the TIM22 mitochondrial protein import complex.

Dihydrolipoic acid reduces cytochrome b561 proteins

Cytochrome b561 (Cyt-b561) proteins constitute a family of trans-membrane proteins that are present in a wide variety of organisms. Two of their characteristic properties are the reducibility by ascorbate (ASC) and the presence of two distinct b-type hemes localized on two opposite sides of the membrane. Here we show that the tonoplast-localized and the putative tumor suppressor Cyt-b561 proteins can be reduced by other reductants than ASC and dithionite. A detailed spectral analysis of the ASC-dependent and dihydrolipoic acid (DHLA)-dependent reduction of these two Cyt-b561 proteins is also presented. Our results are discussed in relation to the known antioxidant capability of DHLA as well as its role in the regeneration of other antioxidant compounds of cells. These results allow us to speculate on new biological functions for the trans-membrane Cyt-b561 proteins.

Heme Ligand Identification and Redox Properties of the Cytochrome c Synthetase, CcmF

Cytochrome c maturation in many bacteria, archaea, and plant mitochondria involves the integral membrane protein CcmF, which is thought to function as a cytochrome c synthetase by facilitating the final covalent attachment of heme to the apocytochrome c. We previously reported that the E. coli CcmF protein contains a b-type heme that is stably and stoichiometrically associated with the protein and is not the heme attached to apocytochrome c. Here, we show that mutation of either of two conserved transmembrane histidines (His261 or His491) impairs stoichiometric b-heme binding in CcmF and results in spectral perturbations in the remaining heme. Exogeneous imidazole is able to correct cytochrome c maturation for His261 and His491 substitutions with small side chains (Ala or Gly), suggesting that a "cavity" is formed in these CcmF mutants in which imidazole binds and acts as a functional ligand to the b-heme. The results of resonance Raman spectroscopy on wild-type CcmF are consistent with a hexacoordinate low-spin b-heme with at least one endogeneous axial His ligand. Analysis of purified recombinant CcmF proteins from diverse prokaryotes reveals that the b-heme in CcmF is widely conserved. We have also determined the reduction potential of the CcmF b-heme (E(m,7) = -147 mV). We discuss these results in the context of CcmF structure and functions as a heme reductase and cytochrome c synthetase.

On the mode of integration of the thylakoid membrane protein cytochrome b(6) into cytoplasmic membrane of Escherichia coli.

In the stroma compartment, several pathways are used for integration/translocation of chloroplast proteins into or across the thylakoid membrane. In this study we investigated the mode of incorporation of the chloroplast-encoded cytochrome b(6) into the bacterial membrane. Cytochrome b(6) naturally comprises of four transmembrane helices (A,B,C,D) and contains two b-type hemes. In the present study, mature cytochrome b(6) or constructed deletion mutants of cytochrome were expressed in E. coli cells. The membrane insertion of cytochrome b(6) in this bacterial model system requires an artificially added presequence that directs the protein to use an E. coli membrane-insertion pathway. This could be accomplished by fusion to maltose-binding protein (MBP) or to the bacterial Sec-dependent signal peptide (SSpelB). The integration of mature cytochrome b(6) into the bacterial cytoplasmic membrane by the Sec pathway has been reported previously by our group (Kroliczewski et al., 2005, Biochemistry, 44: 7570). The results presented here show that cytochrome b(6) devoid of the first helix A can be inserted into the membrane, as can the entire ABCD. On the other hand, the construct devoid of helices A and B is translocated through the membrane into the periplasm without any effective insertion. This suggests the importance of the membrane-anchoring sequences that are likely to be present in only the A and B part, and it is consistent with the results of computational prediction which did not identify any membrane-anchoring sequences for the C or D helices. We also show that the incorporation of hemes into the truncated form of cytochrome b(6) is possible, as long as the B and D helices bearing axial ligands to heme are present.

Catalytic intermediates of cytochrome bd terminal oxidase at steady-state: Ferryl and oxy-ferrous species dominate

The cytochrome bd ubiquinol oxidase from Escherichia coli couples the exergonic two-electron oxidation of ubiquinol and four-electron reduction of O 2 to 2H 2O to proton motive force generation by transmembrane charge separation. The oxidase contains two b-type hemes ( b 558 and b 595) and one heme d, where O 2 is captured and converted to water through sequential formation of a few intermediates. The spectral features of the isolated cytochrome bd at steady-state have been examined by stopped-flow multiwavelength absorption spectroscopy. Under turnover conditions, sustained by O 2 and dithiothreitol (DTT)-reduced ubiquinone, the ferryl and oxy-ferrous species are the mostly populated catalytic intermediates, with a residual minor fraction of the enzyme containing ferric heme d and possibly one electron on heme b 558. These findings are unprecedented and differ from those obtained with mammalian cytochrome c oxidase, in which the oxygen intermediates were not found to be populated at detectable levels under similar conditions [M.G. Mason, P. Nicholls, C.E. Cooper, The steady-state mechanism of cytochrome c oxidase: redox interactions between metal centres, Biochem. J. 422 (2009) 237–246]. The data on cytochrome bd are consistent with the observation that the purified enzyme has the heme d mainly in stable oxy-ferrous and ferryl states. The results are here discussed in the light of previously proposed models of the catalytic cycle of cytochrome bd.