Cytochrome Bo Research Articles

Redox enzymes in tethered membranes.

An electrode surface is presented that enables the characterization of redox-active membrane enzymes in a native-like environment. An ubiquinol oxidase from Escherichia coli, cytochrome bo(3) (cbo(3)), has been co-immobilized into tethered bilayer lipid membranes (tBLMs). The tBLM is formed on gold surfaces functionalized with cholesterol tethers which insert into the lower leaflet of the membrane. The planar membrane architecture is formed by self-assembly of proteoliposomes, and its structure is characterized by surface plasmon resonance (SPR), electrochemical impedance spectroscopy (EIS), and tapping-mode atomic force microscopy (TM-AFM). The functionality of cbo(3) is investigated by cyclic voltammetry (CV) and is confirmed by the catalytic reduction of oxygen. Interfacial electron transfer to cbo(3) is mediated by the membrane-localized ubiquinol-8, the physiological electron donor of cbo(3). Enzyme coverages observed with TM-AFM and CV coincide (2-8.5 fmol.cm(-)(2)), indicating that most-if not all-cbo(3) on the surface is catalytically active and thus retains its integrity during immobilization.

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).

Transcriptional and metabolic response of recombinant Escherichia coli to spatial dissolved oxygen tension gradients simulated in a scale‐down system

Escherichia coli, expressing recombinant green fluorescent protein (GFP), was subjected to dissolved oxygen tension (DOT) oscillations in a two-compartment system for simulating gradients that can occur in large-scale bioreactors. Cells were continuously circulated between the anaerobic (0% DOT) and aerobic (10% DOT) vessels of the scale-down system to mimic an overall circulation time of 50 s, and a mean residence time in the anaerobic and aerobic compartments of 33 and 17 s, respectively. Transcription levels of mixed acid fermentation genes (ldhA, poxB, frdD, ackA, adhE, pflD, and fdhF), measured by quantitative RT-PCR, increased between 1.5- to over 6-fold under oscillatory DOT compared to aerobic cultures (constant 10% DOT). In addition, the transcription level of fumB increased whereas it decreased for sucA and sucB, suggesting that the tricarboxylic acid cycle was functioning as two open branches. Gene transcription levels revealed that cytrochrome bd, which has higher affinity to oxygen but lower energy efficiency, was preferred over cytochrome bO3 in oscillatory DOT cultures. Post-transcriptional processing limited heterologous protein production in the scale-down system, as inferred from similar gfp transcription but 19% lower GFP concentration compared to aerobic cultures. Simulated DOT gradients also affected the transcription of genes of the glyoxylate shunt (aceA), of global regulators of aerobic and anaerobic metabolism (fnr, arcA, and arcB), and other relevant genes (luxS, sodA, fumA, and sdhB). Transcriptional changes explained the observed alterations in overall stoichiometric and kinetic parameters, and production of ethanol and organic acids. Differences in transcription levels between aerobic and anaerobic compartments were also observed, indicating that E. coli can respond very fast to intermittent DOT conditions. The transcriptional responses of E. coli to DOT gradients reported here are useful for establishing rational scale-up criteria and strain design strategies for improved culture performance at large scales.

Redox-induced Protein Structural Changes in Cytochrome bo Revealed by Fourier Transform Infrared Spectroscopy and [13C]Tyr Labeling

Cytochrome bo is a heme-copper terminal ubiquinol oxidase of Escherichia coli under highly aerated growth conditions. Tyr-288 present at the end of the K-channel forms a Cepsilon-Nepsilon covalent bond with one of the Cu(B) ligand histidines and has been proposed to be an acid-base catalyst essential for the O-O bond cleavage at the Oxy-to-P transition of the dioxygen reduction cycle (Uchida, T., Mogi, T., and Kitagawa, T. (2000) Biochemistry 39, 6669-6678). To probe structural changes at tyrosine residues, we examined redox difference Fourier transform infrared difference spectra of the wild-type enzyme in which either L-[1-13C]Tyr or L-[4-13C]Tyr has been biosynthetically incorporated in the tyrosine auxotroph. Spectral comparison between [1-13C]Tyr-labeled and unlabeled proteins indicated that substitution of the main chain carbonyl of a Tyr residue(s) significantly affected changes in the amide-I (approximately 1620-1680 cm(-1)) and -II ( approximately 1540-1560 cm(-1)) regions. In contrast, spectral comparison between [4-13C]Tyr-labeled and unlabeled proteins showed only negligible changes, which was the case for both the pulsed and the resting forms. Thus, protonation of an OH group of tyrosines including Tyr-288 in the vicinity of the heme o-Cu(B) binuclear center was not detected at pH 7.4 upon full reduction of cytochrome bo. Redox-induced main chain changes at a Tyr residue(s) are associated with structural changes at Glu-286 near the binuclear metal centers and may be related to switching of the K-channel operative at the reductive phase to D-channel at the oxidative phase of the dioxygen reduction cycle via conformational changes in the middle of helix VI.

Photothermal studies of CO photodissociation from mixed valence Escherichia coli cytochrome bo3

Here, we report the volume and enthalpy changes accompanying CO photodissociation from the mixed valence form of cytochrome bo 3 oxidase from Escherichia coli. The results of photoacoustic calorimetry indicate two kinetic phases with distinct volume and enthalpy changes accompanying CO photodissociation from heme o 3 and its transfer to Cu B. The first phase occurring on a timescale of <50 ns is characterized by a volume decrease of −1.3 ± 0.3 mL mol −1 and enthalpy change of 32 ± 1.6 kcal mol −1. Subsequently, a volume increase of 2.9 mL mol −1 with an enthalpy change of −5.3 ± 2.5 kcal mol −1 is observed with the lifetime of ∼250 ns (this phase has not been detected in previous optical studies). These volume and enthalpy changes differ from the volume and enthalpy changes observed for CO dissociation from fully reduced cytochrome bo 3 oxidase indicating that the heme o 3/Cu B active site dynamics are affected by the redox state of heme b.

Evidence that Na +-pumping occurs through the D-channel in Vitreoscilla cytochrome bo

The operon ( cyo) encoding the Na +-pumping respiratory terminal oxidase (cytochrome bo) of the bacterium Vitreoscilla was transformed into Escherichia coli GV100, a deletion mutant of cytochrome bo. This was done for the wild type operon and five mutants in three conserved Cyo subunit I amino acids known to be crucial for H + transport in the E. coli enzyme, one near the nuclear center, one in the K-channel, and one in the D-channel. CO-binding, NADH and ubiquinol oxidase, and Na +-pumping activities were all substantially inhibited by each mutation. The wild type Vitreoscilla cytochrome bo can pump Na + against a concentration gradient, resulting in a transmembrane concentration differential of 2–3 orders of magnitude. It is proposed that Vitreoscilla cytochrome bo pumps four Na + through the D-channel to the exterior and transports four H + through the K-channel for the reduction of each O 2.

Role of Tyr-288 at the Dioxygen Reduction Site of Cytochrome bo Studied by Stable Isotope Labeling and Resonance Raman Spectroscopy

To explore the role of a cross-link between side chains of Tyr-288 and His-284 at the heme-copper binuclear center, we prepared cytochrome bo where d(4)-Tyr, 1-[(13)C]Tyr, or 4-[(13)C]Tyr has been biosynthetically incorporated. Unexpectedly, the d(4)-Tyr-labeled enzyme showed a large decrease in the ubiquinol-1 oxidase and CO binding activities. Optical absorption and resonance Raman spectra identified the defect in the distal side of the heme-copper binuclear center. In the CO-bound d(4)-Tyr-labeled enzyme, a large fraction of the nu((Fe-C)) mode was shifted from the normal 520-cm(-1) band to a broad band centered around 491 cm(-1), as found for the Y288F mutant. Our results suggested that the substitution of ring hydrogens of Tyr-288 with deuteriums slows down the formation of the His-Tyr cross-link essential for dioxygen reduction at the binuclear center.

Resonance Raman detection of the Fe2+-C-N modes in heme-copper oxidases: a probe of the active site.

Resonance Raman spectroscopy has been employed to investigate the reduced cyano complexes of cytochrome aa(3) from bovine heart and Rhodobacter sphaeroides and of cytochrome bo(3) from E. coli. In the aa(3)-type oxidases, the frequency of the Fe-CN stretching mode is located at 468 cm(-1), and the bending Fe-C-N vibration, at 500 cm(-1). The fully reduced cytochrome bo(3)-CN complex gives rise to a stretching vibration at 468 cm(-1), a bending vibration at 491 cm(-1), and a stretching C-N vibration at 2037 cm(-1). The observed differences between aa(3) and bo(3) oxidases in the frequencies of the Fe-C-N group suggest a quantitative difference in the structure of the His-heme a(3)(2+)/Cu(B)(1+) and His-heme o(3)(2+)/Cu(B)(1+) binuclear pockets upon CN- binding.

Noninvasive Auto-Photoreduction Used as a Tool for Studying Structural Changes in Heme-Copper Oxidases by FTIR Spectroscopy

We demonstrate an efficient Fourier transform infrared (FTIR) spectroscopic method, termed “auto-photoreduction,” that uses anaerobic photo-induced internal electron transfer to monitor reaction-initiated changes of heme-copper oxidases. It can be applied without the use of either expensive electrochemical equipment, or caged compounds, which cause significant background signals. At high irradiation power, carbon monoxide is released from high-spin heme a of cytochrome c oxidase and heme o from cytochrome bo 3. Photochemistry is initiated at wavelengths <355 nm, and the photochemical action spectrum has a maximum of 290 nm for cytochrome bo 3, which is consistent with the possible intermediate involvement of tyrosinate or an activated state of tyrosine. We propose that the final electron donors are proton channel water molecules. In the pH range of 4–9, the noninvasive auto-photoreduction method yields highly reproducible FTIR redox difference spectra within a broad range, resolving a number of vibrational changes outside the amide I region (1600–1640 cm −1). Furthermore, it provides details of redox-induced changes in the spectral region between 1600 and 1100 cm −1. The auto-photoreduction method should be universally applicable to heme proteins.

The nature of the exchange coupling between high-spin Fe(III) heme o3 and CuBII in Escherichia coli quinol oxidase, cytochrome bo3: MCD and EPR studies.

Fully oxidized cytochrome bo3 from Escherichia coli has been studied in its oxidized and several ligand-bound forms using electron paramagnetic resonance (EPR) and magnetic circular dichroism (MCD) spectroscopies. In each form, the spin-coupled high-spin Fe(III) heme o3 and CuB(II) ion at the active site give rise to similar fast-relaxing broad features in the dual-mode X-band EPR spectra. Simulations of dual-mode spectra are presented which show that this EPR can arise only from a dinuclear site in which the metal ions are weakly coupled by an anisotropic exchange interaction of J 1 cm-1. A variable-temperature and magnetic field (VTVF) MCD study is also presented for the cytochrome bo3 fluoride and azide derivatives. New methods are used to extract the contribution to the MCD of the spin-coupled active site in the presence of strong transitions from low-spin Fe(III) heme b. Analysis of the MCD data, independent of the EPR study, also shows that the spin-coupling within the active site is weak with J approximately 1 cm-1. These conclusions overturn a long-held view that such EPR signals in bovine cytochrome c oxidase arise from an S' = 2 ground state resulting from strong exchange coupling (J > 10(2) cm-1) within the active site.

Dioxygen reduction by bo-type quinol oxidase from Escherichia coli studied by submillisecond-resolved freeze-quench EPR spectroscopy.

The mechanism of the dioxygen (O(2)) reduction conducted by cytochrome bo-type quinol oxidase was investigated using submillisecond-resolved freeze-quench EPR spectroscopy. The fully reduced form of the wild-type enzyme (WT) with the bound ubiquinone-8 at the high-affinity quinone-binding site was mixed with an O(2)-saturated solution, and the subsequent reaction was quenched at different time intervals from 0.2 to 50 ms. The EPR signals derived from the binuclear center and heme b were weak in the time domain from 0.2 to 0.5 ms. The signals derived from the ferric heme b and hydroxide-bound ferric heme o increased simultaneously after 1 ms, indicating that the oxidation of heme b is coupled to the formation of hydroxy heme o. In contrast, the enzyme without the bound ubiquinone-8 (Delta UbiA) showed the faster oxidation of heme b and the slower formation of hydroxy heme o than WT. It is interpreted that the F(I) intermediate possessing ferryl-oxo heme o, cupric Cu(B), and ferric heme b is converted to the F(II) intermediate within 0.2 ms by an electron transfer from the bound ubiquinonol-8 to ferric heme b. The conversion of the F(II) intermediate to the hydroxy intermediate occurred after 1 ms and was accompanied by the one-electron transfer from heme b to the binuclear center. Finally, it is suggested that the hydroxy intermediate possesses no bridging ligand between heme o and Cu(B) and is the final intermediate in the turnover cycle of cytochrome bo under steady-state conditions.

Proton NMR study of the heme environment in bacterial quinol oxidases

The heme environment and ligand binding properties of two relatively large membrane proteins containing multiple paramagnetic metal centers, cytochrome bo 3 and bd quinol oxidases, have been studied by high field proton nuclear magnetic resonance (NMR) spectroscopy. The oxidized bo 3 enzyme displays well-resolved hyperfine-shifted 1H NMR resonance assignable to the low-spin heme b center. The observed spectral changes induced by addition of cyanide to the protein were attributed to the structural perturbations on the low-spin heme (heme b) center by cyanide ligation to the nearby high-spin heme (heme o) of the protein. The oxidized bd oxidase shows extremely broad signals in the spectral region where protons near high-spin heme centers resonate. Addition of cyanide to the oxidized bd enzyme induced no detectable perturbations on the observed hyperfine signals, indicating the insensitive nature of this heme center toward cyanide. The proton signals near the low-spin heme b 558 center are only observed in the presence of 20% formamide, consistent with a critical role of viscosity in detecting NMR signals of large membrane proteins. The reduced bd protein also displays hyperfine-shifted 1H NMR signals, indicating that the high-spin heme centers (hemes b 595 and d) remain high-spin upon chemical reduction. The results presented here demonstrate that structural changes of one metal center can significantly influence the structural properties of other nearby metal center(s) in large membrane paramagnetic metalloproteins.

Fusion protein system designed to provide color to aid in the expression and purification of proteins in Escherichia coli

We have designed and constructed a new fusion expression vector (pKW32), which contains the His-tagged Vitreoscilla hemoglobin (VHb) coding gene upstream of the multiple cloning site. The pKW32 vector was designed to express target proteins as VHb fusions, which can be purified in one step by affinity chromatography. Due to the color of the heme in VHb, the VHb-fused target proteins have a red color that provides a visual aid for estimating their expression level and solubility. The red color can also be used as a visual marker throughout purification, while the concentration of the fusion protein can be determined by measuring the amount of VHb using carbon monoxide difference spectra. In addition, because of inherently high solubility of VHb, the fusion can increase the solubility of sparingly soluble target proteins. Target proteins can be easily separated from His-tagged VHb due to the presence of a thrombin-cleavage site between them. A mutant VHb, the soluble domain of Vitreoscilla cytochrome bo subunit II, and HIV integrase expressed and purified using the pKW32 system have native function. In addition, the integrase, which is known to be difficult to purify because of low solubility, was purified simply and without solubilizing agents using our system.

Purification and Characterization of the MQH2:NO Oxidoreductase from the Hyperthermophilic Archaeon Pyrobaculum aerophilum

The membrane-bound NO reductase from the hyperthermophilic denitrifying archaeon Pyrobaculum aerophilum was purified to homogeneity. The enzyme displays MQH2:NO oxidoreductase (qNOR) activity, consists of a single subunit, and contains heme and nonheme iron in a 2:1 ratio. The combined results of EPR, resonance Raman, and UV-visible spectroscopy show that one of the hemes is bis-His-coordinated low spin (gz = 3.015; gy = 2.226; gx = 1.45), whereas the other heme adopts a high spin configuration. The enzyme also contains one nonheme iron center, which in the oxidized enzyme is antiferromagnetically coupled to the high spin heme. This binuclear high spin heme/nonheme iron center is EPR-silent and the site of NO reduction. The reduced high spin heme is bound to a neutral histidine and can bind CO to form of a low spin complex. The oxidized high spin heme binds NO, yielding a ferric nitrosyl complex, the intermediate causing the commonly found substrate inhibition in NO reductases (Ki(NO) = 7 microm). The qNOR as present in the membrane is, in contrast to the purified enzyme, quite thermostable, incubation at 100 degrees C for 86 min leading to 50% inhibition. The pure enzyme lacks heme b and instead contains stoichiometric amounts of hemes Op1 and Op2, ethenylgeranylgeranyl and hydroxyethylgeranylgeranyl derivatives of heme b, respectively. The archaeal qNOR is the first example of a NO reductase, which contains modified hemes reminiscent of cytochrome bo3 and aa3 oxidases. This report is the first describing the purification and structural and spectroscopic properties of a thermostable NO reductase.

The observation of the dioxygen activation of cytochrome bo from Escherichia coli by submillisecond-resolved freeze quench EPR spectroscopy

The observation of the dioxygen activation of cytochrome bo from Escherichia coli by submillisecond-resolved freeze quench EPR spectroscopy

Kinetics of Intramolecular Electron Transfer in Cytochrome bo3 from Escherichia coli

We have examined the temperature dependence of the intramolecular electron transfer (ET) between heme b and heme o 3 in CO-mixed valence cytochrome bo 3 (C bo) from Escherichia coli. Upon photolysis of CO-mixed valence C bo rapid ET occurs between heme o 3 and heme b with a rate constant of 2.2 × 10 5 s −1 at room temperature. The corresponding rate of CO recombination is found to be 86 s −1. From Eyring plots the activation energies for these two processes are found to be 3.4 kcal/mol and 6.7 kcal/mol for the ligand binding and ET reactions, respectively. Using variants of the Marcus equation the reorganization energy ( λ), electronic coupling factor (H AB), and the ET distance were found to be 1.4 ± 0.2 eV, (2 ± 1) × 10 −3 eV, and 9 ± 1 Å, respectively. These values are quite distinct from the analogous values previously obtained for bovine heart cytochrome c oxidase (C cO) (0.76 eV, 9.9 × 10 −5 eV, 13.2 Å). The differences in mechanisms/pathways for heme b/heme o 3 and heme a/heme a 3 ET suggested by the Marcus parameters can be attributed to structural changes at the Cu B site upon change in oxidation state as well as differences in electronic coupling pathways between Heme b and heme o 3.

Substitutions for glutamate 101 in subunit II of cytochrome c oxidase from Rhodobacter sphaeroides result in blocking the proton-conducting K-channel.

Two functional input pathways for protons have been characterized in the heme-copper oxidases: the D-channel and the K-channel. These two proton-conducting channels have different functional roles and have been defined both by X-ray crystallography and by the characterization of site-directed mutants. Whereas the entrance of the D-channel is well-defined as D132(I) (subunit I; Rhodobacter sphaeroides numbering), the entrance of the K-channel has not been clearly defined. Previous mutagenesis studies of the cytochrome bo(3) quinol oxidase from Escherichia coli implicated an almost fully conserved glutamic acid residue within subunit II as a likely candidate for the entrance of the K-channel. The current work examines the properties of mutants of this conserved glutamate in the oxidase from R. sphaeroides (E101(II)I,A,C,Q,D,N,H) and residues in the immediate vicinity of E101(II). It is shown that virtually any substitution for E101(II), including E101(II)D, strongly reduces oxidase turnover (to 8-29%). Furthermore, the low steady-state activity correlates with an inhibition of the rate of reduction of heme a(3) prior to the reaction with O(2). These are phenotypes expected of K-channel mutants. It is concluded that the predominant entry point for protons going into the K-channel of cytochrome oxidase is the surface-exposed glutamic acid E101(II) in subunit II.

Combined transcriptome and proteome analysis of Escherichia coli during high cell density culture.

Combined transcriptome and proteome analysis was carried out to understand metabolic and physiological changes of Escherichia coli during the high cell density cultivation (HCDC). The expression of genes of TCA cycle enzymes, NADH dehydrogenase and ATPase, was up-regulated during the exponential fed-batch period and was down-regulated afterward. However, expression of most of the genes involved in glycolysis and pentose phosphate pathway was up-regulated at the stationary phase. The expression of most of amino acid biosynthesis genes was down-regulated as cell density increased, which seems to be the major reason for the reduced specific productivity of recombinant proteins during HCDC. The expression of chaperone genes increased with cell density, suggesting that the high cell density condition itself can be stressful to the cells. Severe competition for oxygen at high cell density seemed to make cells use cytochrome bd, which is less efficient but has a high oxygen affinity than cytochrome bo(3). Population cell density itself strongly affected the expression of porin protein genes, especially ompF, and hence the permeability of the outer membrane. Expression of phosphate starvation genes was most strongly up-regulated toward the end of cultivation. It was also found that sigma(E) (rpoE) plays a more important role than sigma(S) (rpoS) at the stationary phase of HCDC. These findings should be invaluable in designing metabolic engineering and fermentation strategies for the production of recombinant proteins and metabolites by HCDC of E. coli.

Direct infrared detection of the covalently ring linked His-Tyr structure in the active site of the heme-copper oxidases.

Infrared spectroscopy, isotopic labeling ([(15)N(delta,epsilon)]histidine and ring-deuterated tyrosine), synthetic model studies, and normal mode calculations are employed to search for the spectroscopic signatures of the unique, covalently linked (His N(epsilon)-C(epsilon) Tyr) biring structure in the heme-copper oxidases. The specific enzyme examined is the cytochrome bo(3) quinol oxidase of E. coli. Infrared features of histidine and tyrosine are identified in the frequency regions of imidazole and phenol ring stretching modes (1350-1650 cm(-1)) and C-H and N-H stretching modes as well as overtones and combinations (>3000 cm(-1)). Two of these, at ca. 1480 and 1550 cm(-1), and their combination tones between 3010 and 3040 cm(-1), are definitively identified with the biring structure involving H284 and Y288 in the E. coli enzyme. Studies of a synthetic analogue of the H-Y structure, 4-methylimidazole covalently linked to p-cresol, show that a feature near 1540 cm(-1) is unique to the biring structure and is absent from the infrared spectrum of 4-methylimidazole or p-cresol alone. This feature is readily detectable by infrared difference techniques, and offers a direct spectroscopic probe for potential radical production involving the H-Y structure in the O(2) reduction cycle of the oxidases.

Vitreoscilla Hemoglobin Binds to Subunit I of Cytochrome bo Ubiquinol Oxidases

The bacterium, Vitreoscilla, can induce the synthesis of a homodimeric hemoglobin under hypoxic conditions. Expression of VHb in heterologous bacteria often enhances growth and increases yields of recombinant proteins and production of antibiotics, especially under oxygen-limiting conditions. There is evidence that VHb interacts with bacterial respiratory membranes and cytochrome bo proteoliposomes. We have examined whether there are binding sites for VHb on the cytochrome, using the yeast two-hybrid system with VHb as the bait and testing every Vitreoscilla cytochrome bo subunit as well as the soluble domains of subunits I and II. A significant interaction was observed only between VHb and intact subunit I. We further examined whether there are binding sites for VHb on cytochrome bo from Escherichia coli and Pseudomonas aeruginosa, two organisms in which stimulatory effects of VHb have been observed. Again, in both cases a significant interaction was observed only between VHb and subunit I. Because subunit I contains the binuclear center where oxygen is reduced to water, these data support the function proposed for VHb of providing oxygen directly to the terminal oxidase; it may also explain its positive effects in Vitreoscilla as well as in heterologous organisms.