Advertisement
JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Azarkina, N.
Right arrow Articles by Konstantinov, A. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Azarkina, N.
Right arrow Articles by Konstantinov, A. A.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

J Biol Chem, Vol. 274, Issue 46, 32810-32817, November 12, 1999


A Cytochrome bb'-type Quinol Oxidase in Bacillus subtilis Strain 168*

Natalia AzarkinaDagger , Sergey SiletskyDagger , Vitaliy BorisovDagger , Claes von Wachenfeldt§, Lars Hederstedt§, and Alexander A. KonstantinovDagger

From the Dagger  A. N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow 119899, Russia and the § Department of Microbiology, Lund University, Sölvegatan 12, SE-223 62 Lund, Sweden

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The aerobic respiratory system of Bacillus subtilis 168 is known to contain three terminal oxidases: cytochrome caa3, which is a cytochrome c oxidase, and cytochrome aa3 and bd, which are quinol oxidases. The presence of a possible fourth oxidase in the bacterium was investigated using a constructed mutant, LUH27, that lacks the aa3 and caa3 terminal oxidases and is also deficient in succinate:menaquinone oxidoreductase. The cytochrome bd content of LUH27 can be varied by using different growth conditions. LUH27 membranes virtually devoid of cytochrome bd respired with NADH or exogenous quinol as actively as preparations containing 0.4 nmol of cytochrome bd/mg of protein but were more sensitive to cyanide and aurachin D. The reduced minus oxidized difference spectra of the bd-deficient membranes as well as absorption changes induced by CO and cyanide indicated the presence of a "cytochrome o"-like component; however, the membranes did not contain heme O. The results provide strong evidence for the presence of a terminal oxidase of the bb' type in B. subtilis. The enzyme does not pump protons and combines with CO much faster than typical heme-copper oxidases; in these respects, it resembles a cytochrome bd rather than members of the heme-copper oxidase superfamily. The genome sequence of B. subtilis 168 contains gene clusters for four respiratory oxidases. Two of these clusters, cta and qox, are deleted in LUH27. The remaining two, cydAB and ythAB, encode the identified cytochrome bd and a putative second cytochrome bd, respectively. Deletion of ythAB in strain LUH27 or the presence of the yth genes on plasmid did not affect the expression of the bb' oxidase. It is concluded that the novel bb'-type oxidase probably is cytochrome bd encoded by the cyd locus but with heme D being substituted by high spin heme B at the oxygen reactive site, i.e. cytochrome b558b595b'.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Bacillus subtilis is a Gram-positive aerobic bacterium, although it can grow anaerobically under some conditions (1, 2). The main components of the aerobic respiratory system of B. subtilis are presented in Scheme 1. Several dehydrogenases (4, 5) transfer electrons from the substrates to an intramembrane pool of menaquinone 7 (3). Menaquinol can be oxidized aerobically either directly by quinol oxidases (QOX)1 or by cytochrome c oxidase (COX) via menaquinol:cytochrome c oxidoreductase and membrane-bound cytochromes c (6).


View larger version (19K):
[in this window]
[in a new window]
  		Scheme 1.   Aerobic respiratory system of B. subtilis. MQ-7, menaquinone 7.

The terminal part of the B. subtilis respiratory chain includes at least three oxidases. The major one is an aa3-type QOX (7-9) that is expressed in rich media at all stages of growth. This enzyme belongs to the heme-copper superfamily (e.g. see Ref. 10) and pumps protons (11, 12). It is encoded by the qoxABCD operon (13) and accordingly contains four subunits (polypeptides QoxA, -B, -C, and -D) (14). The caa3-type COX is expressed during growth on nonfermentable substrates such as succinate (7). It is also a typical proton-pumping heme-copper oxidase and is encoded by the ctaCDEF genes (15). Cytochrome bd can be found in B. subtilis cells grown with glucose (11, 16, 19) and is a QOX that does not pump protons (11) (see also Refs. 17 and 18). The cydAB genes of the cydABCD operon encode the two subunits of cytochrome bd (19). The B. subtilis strain 168 genome sequence also contains genes, ythAB, that seem to encode a second cytochrome bd (19, 20). Cytochrome bd has recently been isolated from Bacillus stearothermophilus (21) and Bacillus firmus OF4 (22), but the B. subtilis enzyme has not yet been isolated or characterized in any detail.

In addition to these three identified oxygen-activating cytochrome complexes, there is experimental evidence pointing to the presence of a fourth terminal oxidase in B. subtilis. A heme protein with spectral characteristics of cytochrome o was partly purified from strain W23 (23). It showed N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) and some low cytochrome c oxidase activity that was relatively resistant to cyanide (I50 = 200 µM). Heme O is an intermediate in the synthesis of heme A from heme B and does not accumulate in B. subtilis unless the final step in heme A synthesis is impaired (24). Since the absorption characteristics of hemes O and B are rather similar (25), it is possible that the "cytochrome o" found in strain W23 (23) was in fact a high spin b-type cytochrome. A 17-kDa CO-reactive cytochrome b has been purified from strain 168 of B. subtilis (11). This cytochrome did not exhibit TMPD- or cytochrome c-oxidase activity (<1.5 s-1) and was considered unlikely to be a terminal oxidase. The cytochrome preparations mentioned above were reported to lack quinol oxidase activity. However, this could be due to the nonoptimal quinols used as electron donors. The respiratory enzymes in B. subtilis are known to operate much better with the low potential naphthoquinoles such as dimethylnaphthoquinol (14, 26, 27) than with menadiol or benzoquinol derivatives.

Lemma et al. (14) reported that a mutant of B. subtilis W23 deleted for the qoxB gene and grown with glucose did not contain cytochromes a or d, as determined from optical absorption spectra, but showed significant quinol oxidase activity with dimethylnaphthoquinol as the electron donor. This led the authors to suggest the existence of an additional unidentified QOX in B. subtilis, possibly of the cytochrome o type. Similar observations have been made by Villani et al. (12) with a B. subtilis mutant lacking the entire qoxABCD operon.

We considered it worthwhile to carry out a more systematic investigation of the putative fourth terminal oxidase in B. subtilis. In order to have a convenient experimental model, we constructed a mutant of strain 168, LUH27, lacking the structural genes for aa3-QOX, caa3-COX, and succinate:menaquinone oxidoreductase. Deficiency in the two aa3-type terminal oxidases was expected not only to eliminate their contribution to respiratory activity and absorption spectra but also to stimulate expression of alternative oxygen-activating enzymes. Succinate:menaquinone oxidoreductase was removed mainly because the diheme cytochrome b558 in this enzyme dominates the absorption spectra of wild type membranes (5). B. subtilis mutants deleted for genes encoding cytochrome bd and aa3-QOX, i.e. cydABCD and qoxABCD, can only be grown anaerobically (28). However, by growing strain LUH27 under different conditions, the content of cytochrome bd in the cells can be varied more than 10-fold, and cells with barely detectable levels of this oxidase can be obtained. This work reports results from our initial studies with strain LUH27.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Chemicals-- Aurachin D was a kind gift of Prof. Peter Rich (University College, London) obtained through the courtesy of Dr. Jeff Osborne (University of Illinois, Urbana-Champain, IL). A CO cylinder ("for synthesis" grade) was purchased from Merck. Other chemicals were commercial products of high purity from conventional sources (Sigma, Serva, Fluka, and Merck).

Construction of Plasmid pYTH1-- The 2.8-kilobase pair ythABC region of the B. subtilis chromosome was amplified by polymerase chain reaction using DNA from strain 1A1 (trpC2) as template, the oligonucleotides 5'-CCGGATCCTTCCCTCTGTTTCACATCGTA-3' (BamHI site underlined) and 5'-GGTCTAGATCTTTTAATTATAAATCATGCGGC-3'(XbaI site underlined), and the Expand high fidelity polymerase chain reaction system (Roche Molecular Biochemicals) according to the manufacturer's protocol. The polymerase chain reaction product was digested with BamHI and XbaI, ligated to pHPSK (29), and used to transform B. subtilis strain 168 (trpC2) to chloramphenicol resistance. The resulting plasmid containing the ythABC genes and flanking regions was named pYTH1.

Construction of Strain LUH27-- B. subtilis strain 168 was transformed to phleomycin resistance with chromosomal DNA from strain JO1, which contains a Delta ctaCD::ble deletion (30). The resulting strain, LUH15 (trpC2 Delta ctaCD::ble), was then transformed to chloramphenicol resistance with strain 3G18Delta 12 DNA containing a Delta sdhCA'::cat mutation (31). One transformant was kept and named LUH19 (trpC2 Delta ctaCD::ble Delta sdhCA'::cat). The triple respiratory deficient mutant LUH27 (trpC2 Delta ctaCD::ble Delta sdhCA'::cat Delta qoxABCD::kan) was obtained by the transformation of strain LUH19 to neomycin resistance with chromosomal DNA from a strain containing a Delta qoxABCD::kan mutation (12).

Construction of Strain LUW123-- To construct a ythAB deletion mutant, a 740-base pair HindIII-PvuII fragment of pYTH1, containing part of ythB and ythC, was ligated to pDG1515 (32) that had been digested with XhoI, treated with the large fragment (Klenow) of Escherichia coli DNA polymerase I, and digested with HindIII. The resulting plasmid was named pYTH10. A part of ythA on pYTH1 was excised as a 470-base pair BamHI-PstI fragment and ligated to pYTH10 that had been digested with the same enzymes. The resulting plasmid was used to transform strain 168 to tetracycline resistance, resulting in LUW122. The substitution of the ythAB::tet allele for the wild-type ythAB genes (as a result of double-crossover homologous recombination) in strain LUW122 was confirmed by Southern blot analysis. Strain LUW123 (trpC2 Delta ythAB::tet Delta ctaCD::ble Delta sdhCA::cat Delta qoxABCD::kan) was obtained by transforming LUH27 with chromosomal DNA isolated from LUW122.

Transformation of B. subtilis Strains with Chromosomal or Plasmid DNA-- Transformations were performed essentially as described by Hoch (33), and transformants were selected on tryptose blood agar base plates containing chloramphenicol (4 or 5 mg/liter), phleomycin (3 mg/liter), neomycin (5 mg/liter), or tetracyclin (15 mg/liter) as appropriate.

Growth of Bacteria in Liquid Media-- The yeast extract medium with phosphate (YMP) used in this work is the same as the nutrient broth sporulation medium with phosphate (34) except that the nutrient broth was replaced by 0.5% (w/v) yeast extract (Difco). Where indicated, 0.5% (w/v) glucose was added. The cells were grown at 37 °C on a rotary shaker at 200 rpm in 5-liter indented flasks containing 1 or 2 liters of medium for high or low aeration conditions, respectively. Cultures were harvested at the end of the exponential phase (A600 was 1.5 and 0.4-0.5 for the growth with and without glucose, respectively). In the case of proton pumping experiments, the phosphate concentration in the growth medium was decreased to 5 mM, which was verified not to influence the cytochrome composition of the cells.

Isolation of Membranes-- Membranes were isolated as described before (34) and stored suspended in 20 mM MOPS buffer, pH 7.4, at -70 °C.

Protein Concentrations-- Protein was determined using the BCA protein assay reagent (Pierce).

Heme Analysis-- Heme composition of membranes was assayed by reverse-phase high pressure liquid chromatography (HPLC) as described before (24).

Spectroscopic Assays-- Conventional optical absorption difference spectra were recorded using a SLM-Aminco DW-2000 UV-visible spectrophotometer operating in split beam mode and standard rectangular glass cuvettes with a 10-mm light path. Unless indicated otherwise, the slit width was 3 nm and the scan rate was 2 nm/s. The experiments were performed in M1 medium (100 mM MOPS, 0.2 mM EDTA, pH 7.0) at a final membrane concentration of 1-5 mg of protein/ml. For "reduced minus oxidized" difference spectra, the aerobically preincubated sample was considered to be "fully oxidized." Reduction of the samples was achieved by the addition of a few grains of solid sodium dithionite. An extinction coefficient of Delta epsilon 630-650 = 17 mM-1 cm-1 (52) was used to evaluate the concentration of cytochrome bd in the membranes from the "dithionite reduced minus aerobically oxidized" difference absorption spectra.

Kinetics of CO Recombination-- Flash-induced dissociation of CO and kinetics of its subsequent recombination with membrane-bound cytochromes were measured in a single beam spectrophotometer constructed by Prof. L. A. Drachev at the A. N. Belozersky Institute of Physico-Chemical Biology, Moscow State University. Monitoring light from a 75-watt halogen lamp aligned perpendicular to the excitation beam from the laser was filtered through a grating monochromator and passed through the sample positioned in a thermostated 10 × 10-mm rectangular quartz cell with four optically transparent sides. Upon exiting the sample cuvette, the monitoring light beam was sent via a second monochromator (prism) to a photomultiplier. A glass color filter was placed before the second monochromator in order to absorb scattered laser light. The signal from the photomultiplier, passed through an appropriate RC filter (10 µs to 1 ms depending on the reaction rate), was recorded with a PC-interfaced digital transient recorder (Datalab-1080). CO was added to anaerobic dithionite-reduced membranes as small volumes of CO-saturated buffer with dithionite. Photodissociation of CO was induced by 10-ns light pulses from a neodimium YAG laser Quantel 481 operated in a doubled frequency mode (lambda  = 532 nm). Exponential decay curve fitting was done with the software package GIM (Graphic Interactive Management) developed by A. L. Drachev.

Respiratory Activity and Proton Pumping Assays-- Oxygen consumption was monitored amperometrically with a Clark-type oxygen electrode at 25 °C in M1 medium containing 0.1-0.3 mg/ml of membrane protein. The assay was initiated by the addition of a substrate. In the case of the quinol oxidase assays, dithiothreitol (DTT) and Q2 were added together from a stock mixture in which full reduction of Q2 had been verified spectrophotometrically. Proton pumping was assayed using the "oxygen pulse" method as described in Ref. 12, except that KSCN was replaced by valinomycin.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cytochrome bd Content in Strain LUH27 under Different Growth Conditions-- Fig. 1 shows typical difference absorption spectra (reduced minus oxidized) of membranes from strain LUH27 grown without glucose at high aeration (A) or with glucose at low aeration (B). The membranes do not show any evidence for the presence of heme A-containing oxidases but contain cytochrome bd. Growth of LUH27 with glucose plus oxygen limitation resulted in an approximate 10-fold increase of the cytochrome bd-specific content of the membranes (from about 0.04 to about 0.4 nmol/mg of protein). These two types of preparations are referred to below as bd- and bd+ membranes.


View larger version (13K):
[in this window]
[in a new window]
  		Fig. 1.   Absorption spectra of LUH27 membranes with low (A) and high (B) content of cytochrome bd. The difference spectra (reduced with dithionite minus aerobically oxidized) were recorded as described under "Experimental Procedures." The absorption changes are normalized to 1 mg of protein/ml; the insets show enlarged alpha -band regions of the spectra. C shows the line shapes of the difference spectra in the far red region specific for cytochrome bd contribution. Traces a and b correspond to the full spectra in A and B, respectively; trace a has been expanded 10-fold relative to trace b and smoothed.

The line shapes of the difference spectra of the bd- and bd+ membranes are distinctly different in the far red region (Fig. 1C). First, a trough at about 650 nm typical of the reduction-induced decay of heme D oxycomplex (trace b) is barely visible in the spectrum of the bd- membranes (trace a); this could indicate that heme D in the aerobic bd- membranes is largely in the oxidized rather than the oxygenated state. Second, the difference spectrum of the bd- membranes reveals an approximate 3-fold higher relative contribution (plus some band shift to the red) of the component absorbing around 600 nm (presumably heme b595).

Heme Composition of LUH27 Membranes-- Analysis of the hemes extracted from bd- membranes showed the presence of heme B, as expected, but no heme A or O could be detected (Fig. 2). Therefore, the bd- membranes cannot contain a bo-type terminal oxidase. The results of the heme analysis also show that the absorption maximum at 600 nm in the reduced minus oxidized difference spectrum of the bd- membranes (Fig. 1C, trace a) is not due to heme A and rather belongs to high spin heme b.


View larger version (14K):
[in this window]
[in a new window]
  		Fig. 2.   HPLC analysis of heme in LUH27 membranes with a low content of cytochrome bd. Hemes were eluted from the column by an acetonitrile gradient in water containing 0.05% trifluoroacetic acid and detected by their absorption at 406 nm. The downward arrows indicate positions in the chromatogram at which hemes B, A, and O from control extracts were eluted.

Respiratory Activity of LUH27 Membranes-- Oxygen consumption by bd- and bd+ membranes with different electron donors and the effects of some inhibitors are shown in Table I. The rate of respiration with NADH as the substrate did not depend significantly on the cytochrome bd content. This observation could indicate that a terminal oxidase other than cytochrome bd is responsible for oxygen consumption in the bd- samples.

                              
View this table:
[in this window]
[in a new window]
 
Table I
Respiratory activity of LUH27 bd- membranes
Where indicated, the numbers in parentheses give the activities measured under the same conditions with the bd+ membranes. The concentrations used were as follows: NADH, 0.3 mM; DTT, 5 mM; KCN, 1 mM; aurachin D, 40 µM. The membranes were preincubated with aurachin D for 10 min before the addition of the electron donor.

Quinol oxidase activities of bd- and bd+ membranes were compared using oxidation of DTT as stimulated by Q2. DTT and reduced Q2 are rather poor electron donors for the B. subtilis respiratory system as compared with NADH or to dimethylnaphthoquinol (14, 26), but nevertheless, the reaction is likely to report on the quinol oxidase activity of the membranes. The DTT-supported oxygen consumption was stimulated by Q2, inhibited by cyanide and aurachin D, and abolished by heat inactivation of the membranes. Notably, at the same concentrations of Q2 and DTT, the specific respiratory activities of bd- and bd+ samples were very similar in agreement with the similar NADH-oxidase activities.

Both types of membranes catalyzed oxygen consumption with ascorbate either alone or with mediators such as TMPD and cytochrome c. However, this activity of the preparations is probably of nonenzymatic origin, since it was resistant to cyanide, partly sensitive to EDTA, and could be observed also with boiled membranes.

As shown in Table II, the NADH-dependent respiration of the two types of membranes differ in sensitivity to inhibitors. Oxygen consumption by the bd- membranes was considerably more sensitive to cyanide. Interpolation of the data in the Dixon coordinates (1/v versus [inhibitor]; not shown) gives I50 values of about 120 and 550 µM for the bd- and bd+ membranes, respectively. The bd- membranes were also more sensitive to aurachin D, an antibiotic known to inhibit preferentially bd-QOX, probably at the quinol-binding site (35, 36).

                              
View this table:
[in this window]
[in a new window]
 
Table II
Inhibitor sensitivity of NADH-oxidase activity of LUH27 bd- and bd+ membranes
The basic conditions were the same as in Table I.

CO-induced Absorption Changes of Cytochromes in LUH27 Membranes-- The effect of carbon monoxide on the absorption spectra of dithionite-reduced bd- and bd+ membranes is shown in Fig. 3A. For the bd+ membranes (spectrum b), the CO-induced difference spectrum is typical of cytochrome bd as observed for instance for the E. coli and Azotobacter vinelandii enzymes (37-40). A red shift in the far red region (a trough at 622 nm and a peak at 643 nm) indicates interaction of heme d with CO (40). A broad minimum around 595 nm and a rather deep trough at 444 nm appeared rapidly; these features resemble the spectral response of A. vinelandii cytochrome bd (37, 39) and may indicate CO binding with a fraction of high spin heme b595. A local minimum near 561 nm and an intensive blue shift in the Soret region with a trough at 429 nm and a peak at about 416 nm developed upon prolonged bubbling with CO and may report interaction of the ligand with the low spin heme b558 (38, 41, 42).


View larger version (16K):
[in this window]
[in a new window]
  		Fig. 3.   CO-reactive cytochromes in LUH27 membranes with high and low content of cytochrome bd. A, difference spectra induced by the addition of 1 mM CO to the dithionite-reduced membranes. Spectrum a, bd- membranes; spectrum b, bd+ membranes. See legend to Fig. 1 and "Experimental Procedures" for the conditions. B, concentration dependence of the absorption changes induced by CO in the Soret band of the dithionite-reduced bd- membranes. For each CO concentration, the ligand-induced difference spectrum in the Soret was recorded. The data were obtained at a membrane concentration of 1.6 mg of protein/ml (~0.18 µM concentration of the bb' oxidase) and are normalized to 1 mg of protein/ml. Inset, difference spectrum of the low affinity phase of the CO-induced changes (1 mM CO, 4-min bubbling versus 20 µM CO). C, kinetics of CO recombination in the bd- (trace a, 436 nm) and bd+ (trace b, 445 nm) membranes. The experiments were performed as described under "Experimental Procedures" in buffer containing 100 mM MOPS, pH 7.0, 0.2 mM EDTA, and 10% glycerol (to decrease light scattering by the turbid sample). Membrane concentration was 0.6 mg of protein/ml. The data are normalized to 1 mg of protein/ml. CO concentration was 3 µM. At these low rates of recombination, the data were collected with the 103-Hz RC filter and represent an average of 50 5-s-spaced transients.

The extensive CO-induced absorption changes observed with bd- membranes (Fig. 3A, spectrum a) are very different from those in the bd+ membranes. As expected, there is no significant contribution from cytochrome bd in the far red region. The main feature of the difference spectrum is a blue shift of the Soret band with a maximum at 418 nm and a broad minimum at 433 nm that is accompanied by a minute trough at about 560 nm. This kind of CO-induced absorption change is typical of high spin heme O- or heme B-containing proteins (42) and has been observed for o-type terminal oxidases (43-46) and for high spin heme B-containing enzymes (47-50). Thus, the data suggest the presence of a high spin cytochrome that may be responsible for the respiratory activity of the bd- membranes. Since the heme analysis did not reveal any heme O in the membranes, it is reasonable to denote this oxidase as a bb3 or bb' type enzyme (as recommended by Poole (51)). Assuming an extinction coefficient of about 150 mM-1 cm-1 for the CO-induced difference spectrum in the Soret band (42), the specific content of the bb' oxidase in the bd- membranes is about 0.11 nmol/mg of protein.

The CO-induced absorption changes in the bd- membranes saturate at about 3 µM of the ligand (Fig. 3B). This high affinity phase of titration is fitted well by a theoretical curve for a single binding site with an apparent Kd of 0.45 µM. At micromolar concentrations of CO, the difference spectra did not reveal any discernible changes in the 550-570-nm range (not shown, the gamma /alpha response ratio >= 20), which is diagnostic of CO combination with a high spin heme b (42). In contrast, the time-dependent increase of the ligand-induced response in the gamma -band observed at 1 mM CO was associated with growth of a trough around 560 nm in the difference spectrum with Delta Agamma /Delta A560 of about 10 (Fig. 3B, inset; cf. spectrum a in Fig. 3A). This second phase of absorption changes is likely to be dominated by low affinity binding of CO with the low spin heme b in the bb' oxidase, as observed for purified cytochrome bd from E. coli and A. vinelandii (52) and membrane-bound cytochrome bd of B. subtilis.2

Flash-induced dissociation/recombination of CO in the bd- and bd+ membranes is shown in Fig. 3C. Data obtained for the reaction at a low concentration of CO (3 µM) is shown in order to avoid significant contribution from the low affinity binding of CO with low spin heme b (cf. multiphasic CO recombination with the solubilized cytochrome bd from A. vinelandii (53)). Detailed analysis of CO interaction with the B. subtilis bd- and bb'-type oxidases at different concentrations of the ligand in comparison with E. coli cytochrome bd will be described elsewhere.3 The kinetics of CO recombination with cytochrome bd in the bd+ membranes (Fig. 3C, trace b) was similar to that observed for E. coli cytochrome bd (54). At this low concentration of CO, the trace is practically monophasic (94%) with tau  = 70 µs if recalculated to the conventional 1 mM CO. Recombination of CO in the bd- membranes (Fig. 3C, trace a) was roughly 10-fold slower. The minor initial rapid phase (~20%) is due to a recombination with a fraction of cytochrome bd in the bd- membranes (cf. Fig. 1, A and C), and the rest of the A436 decay is due to rebinding of CO with high spin heme(s) b as shown by the time-resolved spectra of the phases.3 Recombination with heme b was dominated (~70%) by a component with tau  = 2.8 ms (extrapolated to 1 mM CO), but about 30% of the recombination was faster (240 µs at 1 mM CO). On average, CO recombination with B. subtilis cytochrome bb' was about 10-fold faster than that with mitochondrial or bacterial heme-copper oxidases such as cytochrome bo', ba3, or aa3 (typically, tau  = 15-30 ms at 1 mM CO and room temperature (e.g. see Ref. 55), which converts to 5-10 s at 3 µM of CO as used in Fig. 3C).

Cyanide-induced Absorption Changes-- The addition of KCN to the fully reduced bd- membranes gave rise to relatively small absorption changes in the gamma -band, as compared with those induced by CO, with a trough at 428 nm close to the ferrous cytochrome b absorption band and a small increase of absorbance around 411 nm (Fig. 4). Maximum changes occurred in less than 3 min and resembled those observed for cyanide binding with the reduced E. coli cytochrome bo3 (56).


View larger version (28K):
[in this window]
[in a new window]
  		Fig. 4.   Cyanide-induced absorption changes in reduced LUH27 membranes with a low content of cytochrome bd. The difference spectra were recorded at 3 and 32 min after the anaerobic addition of 50 mM KCN to the dithionite-reduced membranes. The traces have been corrected for a base-line drift by subtraction of a straight line connecting the data points at 405 and 444 nm of the difference spectra.

As illustrated by Fig. 5, cyanide is likely to compete with CO for binding with the reduced bb' oxidase. Since the absorption changes induced by cyanide are much smaller than those induced by CO, it is possible to directly observe competition between the two ligands spectrophotometrically. Preincubation of the reduced bd- membranes with 50 mM KCN greatly diminished the response induced by a low (1.6 µM) concentration of CO (Fig. 5A). A high concentration of CO overcomes the effect of cyanide and results in a response that is about 70% of the control (cf. spectra b and a in Fig. 5B). The antagonistic effects of KCN and CO are consistent with competition between the two ligands for binding at the same site, presumably with high spin heme b.


View larger version (15K):
[in this window]
[in a new window]
  		Fig. 5.   Prevention by cyanide of the CO-induced absorption changes in the reduced LUH27 membranes with a low content of cytochrome bd. The figure shows the difference spectra induced by low (1.6 µM, A) and high (1 mM, B) concentrations of CO in the dithionite-reduced membranes. In traces b (scanned at 0.5 nm/s), the reduced membranes had been preincubated anaerobically with 50 mM KCN before the addition of CO. Traces a are the controls without KCN. In A, CO was added as a small volume of the anaerobic buffer saturated with CO. In B, CO was bubbled through the sample. Other conditions were as in Fig. 1.

Reaction of cyanide with bd+ and bd- membranes under oxidizing conditions is shown in Fig. 6. In the bd+ membranes, the absorption changes induced by cyanide developed rapidly, reaching saturation in less than 1 min (Fig. 6A). The difference spectra closely resemble those observed for the "aerobically oxidized" ("as isolated") E. coli cytochrome bd (57, 58). The trough at 650 nm originates from cyanide-induced decay of the oxycomplex of heme d. The concomitant red shift in the gamma -band (trough at 410 nm, peak at 438 nm) is very similar to those observed for the purified E. coli cytochrome bd (a trough at 408 nm and a peak at 437 nm) in parallel with the cyanide-induced decomposition of the oxycomplex (cf. Fig. 3A in Ref. 57). This red shift is probably dominated by cyanide-induced changes of hemes b (41, 57-59).


View larger version (23K):
[in this window]
[in a new window]
  		Fig. 6.   Cyanide-induced absorption changes in aerobically oxidized LUH27 membranes with a high (A) and low (B) content of cytochrome bd. The difference absorption spectra were scanned at the indicated moments after the addition of 5.2 mM NaCN to the air-oxidized membranes (the samples were stirred aerobically for 30 min before the addition of cyanide); time corresponds to beginning of the scan at 380 nm. The flat featureless long wavelength parts of the difference spectra in the case of the bd- membranes (B) are omitted. See Fig. 1 and "Experimental Procedures" for details.

The absorption changes induced by cyanide in the gamma -band of the bd- membranes under oxidizing conditions are shown in Fig. 6B and are quite different from those in the bd+ preparation (Fig. 6A). The addition of 5 mM cyanide to the aerobic bd- membranes resulted in a red shift of the Soret band with no evidence for a trough at 650 nm. A derivative-shaped difference spectrum with minimum at 408 nm and maximum at 435 nm developed slowly and reached saturation in about half an hour. The peak-to-trough size of the response is about 50 mM-1 cm-1 (assuming the specific content of the bb' oxidase in the sample to be 0.11 nmol/mg of protein; see above). These changes were preceded by the appearance of an asymmetric peak at ~423 nm in the first 3 min, presumably due to some reduction of the heme(s) by endogenous electron donors. The cyanide-induced red shift of the gamma -band in the oxidized bd- membranes is similar to that observed for the oxidized E. coli cytochrome bo3 (60), except for a shift of the entire difference spectrum to longer wavelengths (peak/trough at 408/435 nm rather than at 401/422 nm as in Ref. 60). This shift is consistent with different positions of the Soret bands of the high spin cytochromes b3+ and o3+ (61).

Of other ferric heme iron ligands tested, H2O2 added to the oxidized bd- membranes at 100 µM resulted in instantaneous Soret band absorption changes with a minimum at about 412 nm and a maximum at 430-440 nm, typical of a red shift of the gamma -absorption band.

Proton Pumping-- Fig. 7 shows acidification of the medium induced by oxygenation of LUH27 bd- and bd+ cells and LUW10 cells. Strain LUW10 contains the proton-pumping oxidases aa3-QOX and caa3-COX but lacks the nonpumping cytochrome bd (19). Proton release by LUW10 cells is characterized by a H+/e- ratio close to 2, whereas in case of the LUH27 bd+ and bd- cells, the H+/e- ratio observed is about half. These data corroborate results obtained by Villani et al. (12) with B. subtilis cells devoid of the aa3-QOX and indicate that the bb' oxidase, like cytochrome bd, does not pump protons.


View larger version (15K):
[in this window]
[in a new window]
  		Fig. 7.   Proton pumping by LUW10 and LUH27 cells. Fresh cells were spun down rapidly and resuspended in 100 mM KCl, 1 mM MgSO4 at a concentration of 5-6 OD units at 600 nm. The cells depleted oxygen rapidly in the closed reaction vessel, and pH attained a value of 6-6.3 in 2-3 min and then remained constant. Acidification of the medium in response to small additions of oxygen (5 µM) injected with water (indicated by arrows) was followed with a glass pH electrode. Proton efflux was greatly facilitated in the presence of valinomycin (1 µM), which discharges the cell membrane (note the control trace (without valinomycin)). The response was calibrated by the addition of small volumes of 20 mM HCl, and the amount of H+ extruded was compared with the amount of consumed oxygen. The H+/e- values were 1.8, 0.75, and 0.77 for LUW10, LUH27 bd+, and LUH27 bd- cells, respectively.

The bb' Oxidase Is Independent of ythAB-- To determine if the bb' oxidase is encoded by the ythABC gene cluster, ythA and ythB were deleted from the chromosome of strain LUH27, resulting in strain LUW123 (see "Experimental Procedures"). The cytochrome composition of LUW123, and also LUH27 containing the ythABC genes on plasmid (pYTH1), grown in YMP with or without glucose added were similar to those of LUH27 (Table III). Membrane preparations from LUH27/pYTH1 grown with glucose, however, consistently contained more cytochrome bd as compared with the corresponding membranes from LUH27 and LUW123. Importantly, the CO-induced difference absorption spectra of the bd- membranes from LUW123 and LUH27/pYTH1 were similar in size and line shape to those of LUH27 (spectra not shown). Furthermore, the CO-induced spectra were fully saturated at 3 µM CO, indicating high affinity binding with a high spin b-type cytochrome. These results demonstrate that the bb'-type oxidase is not encoded by the ythAB genes.

                              
View this table:
[in this window]
[in a new window]
 
Table III
Cytochrome content of membranes from different strains grown in YMP with glucose at low aeration (bd+) and without glucose at high aeration (bd-)
The cytochrome content was determined from the dithionite reduced minus air-oxidized difference spectra of membranes using extinction coefficients of 17 mM-1 cm-1 for cytochrome bd (Delta A at 628-655 nm), and 20 mM-1 cm-1 for cytochrome b (Delta A at 563-575 nm). The values shown are the average from the analysis of 2-4 preparations.

The respiratory activities of membranes from LUW123, LUH27/pYTH1, and LUH27 were very similar with NADH or Q2/DTT as substrates (data not shown). Cytochrome bd+ membranes of LUW123 and LUH27/pYTH1 showed higher resistance of respiration to cyanide than bd- membranes as observed for LUH27 membranes.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

A Novel Oxidase of bb' Type in B. subtilis Strain 168-- This work provides substantial evidence for the presence of a fourth terminal oxidase in B. subtilis 168 in addition to the previously identified aa3-QOX, caa3-COX, and bd-QOX oxidases. Our experiments have led to two basic findings. First, bacteria lacking the structural genes for both the heme A-containing oxidases can grow aerobically, and the respiratory activity is virtually independent of the cytochrome bd content. Second, the absence of the succinate:menaquinone oxidoreductase-associated cytochrome b in strain LUH27 has allowed us to detect unambiguously the presence of a high spin b-type cytochrome in the bd- membranes that reacts with CO, cyanide, and hydrogen peroxide. We assume provisionally that these observations are related and that the membranes from LUH27 cells with a low content of cytochrome bd contain an additional terminal oxidase that is responsible for the ligand-induced absorption changes and contributes to cell growth and respiration. According to the optical absorption characteristics and the results of heme analysis, this new oxidase contains heme B only and should be denoted as an oxidase of a bb' type.

The presence of bb'-type oxidases may be common for Bacillus species as indicated from analyses of other organisms (62-65). A similar type of oxidase has been described for Pseudomonas nautica 617 (66), P. aeruginosa (67), and Rhodobacter sphaeroides (68).

We could not find cytochrome c-oxidase or TMPD-oxidase activities in bd- membranes of strain LUH27, but these membranes showed quinol oxidase activity. Similar to the "cytochrome o-like" hemoprotein described by de Vrij et al. (23) and the putative novel oxidase reported by Villani et al. (12), the bb'-QOX in LUH27 is relatively resistant to inhibition by cyanide (50% inhibition at about 120 µM) but is inhibited by aurachin D. These properties resemble those of cytochromes bd (reviewed in Ref. 52). It is noteworthy that according to our data and results of Villani et al. (12), this oxidase does not pump protons, which is also typical of bd-type quinol oxidases as opposed to the oxidases of the heme-copper superfamily (52). In addition, the new oxidase binds CO much faster than is typical of heme-copper oxidases, although still slower than the bd-type oxidase in the same strain. It is notable that P. aeruginosa contains a terminal oxidase of the bd type seemingly operating in the absence of heme D (67).

Which B. subtilis Genes Encode the Novel Oxidase?-- The complete sequence of the B. subtilis genome (20) reveals structural genes for four oxidases (see the Subtilist data base on the Internet). Three of these are the known oxidases, namely aa3-QOX, caa3-COX, and bd-QOX. The fourth is a putative second cytochrome bd encoded by the ythABC gene cluster. The role of this gene cluster in B. subtilis has not yet been established, but we show in this work that the ythAB genes are not required for growth or for the synthesis of the bb' oxidase.

It is known that bacteria can incorporate different hemes in both the low spin and high spin sites of the same terminal oxidase protein (69, 70-72). For example, a bb3 variant of ba3-QOX from Paracoccus denitrificans (although enzymatically inactive) has been described (73) in which heme B substitutes for heme A in the binuclear center as a result of impaired heme A biosynthesis. It is thus possible that the novel B. subtilis oxidase corresponds to the CydAB polypeptides containing three hemes B: one low spin (b558) and two high spin (b595 and b'). The presence of heme b595 in the bb' oxidase might explain the abnormally high A600/A628 ratio observed in the "bd-specific" far red region of the reduced minus oxidized spectrum of the LUH27 bd- membranes (Fig. 1C). Circumstantial evidence in favor of heme b595 being present in the bb' oxidase is also provided by the CO-induced difference spectra. At micromolar concentrations of the ligand, the CO-induced trough at 434 nm is clearly accompanied by a shoulder at 444 nm (Fig. 5A, spectrum a), diagnostic of CO binding with heme b595 (41, 74).

We conclude that the bb'-type oxidase in B. subtilis is either encoded by some of the many genes of not yet assigned function or is the cytochrome bd, which under some conditions, and for unknown reasons, incorporates heme B instead of heme D in the oxygen-reducing site.

    ACKNOWLEDGEMENTS

We thank Lena Winstedt for help in running the polymerase chain reaction and Dr. Nick. E. Le Brun for linguistic advice.

    FOOTNOTES

* This work was supported in part by Russian Fund for Basic Research Grants 97-04-49765 (to N. A., A. A. K., and S. S.), 98-04-48847 (to N. A., S. S., and V. B.), and 99-04-48095 (to N. A. and V. B) and by grants from INTAS-RFBR 95-1259 (to A. A. K. and C. von W.), the Swedish Royal Academy of Sciences (to L. H. and A. A. K.), and the Swedish Natural Science Research Council (to L. H.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Dept. of Microbiology, Lund University, Sölvegatan 12, SE-22362 Lund, Sweden. Tel.: 46-46-2228622; Fax: 46-46-157839; E-mail: Lars.Hederstedt@ mikrbiol.lu.se.

2 N. Azarkina and A. A. Konstantinov, unpublished data.

3 N. Azarkina and A. A. Konstantinov, manuscript in preparation.

    ABBREVIATIONS

The abbreviations used are: QOX, quinol oxidase(s); COX, cytochrome c oxidase; DTT, dithiothreitol; MOPS, 3-(N-morpholino)propanesulfonic acid; HPLC, high pressure liquid chromatography; Q2, decylubiquinone; TMPD, N,N,N',N'-tetramethyl-p-phenylenediamine.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. Hoffmann, T., Frankenberg, N., Marino, M., and Jahn, D. (1998) J. Bacteriol. 180, 186-189[Abstract/Free Full Text]
2. Nakano, M. M., Dailly, Y. P., Zuber, P., and Clark, D. P. (1997) J. Bacteriol. 179, 6749-6755[Abstract/Free Full Text]
3. Collins, M. D., and Jones, D. (1981) Microbiol. Rev. 45, 316-354[Free Full Text]
4. Bergsma, J., van Dongen, M. B. M., and Konings, W. N. (1982) Eur. J. Biochem. 128, 151-157[Medline] [Order article via Infotrieve]
5. Hägerhäll, C., Aasa, R., von Wachenfeldt, C., and Hederstedt, L. (1992) Biochemistry 31, 7411-7421[CrossRef][Medline] [Order article via Infotrieve]
6. Yu, J, Hederstedt, L., and Piggot, P. J. (1995) J. Bacteriol. 177, 6751-6760[Abstract/Free Full Text]
7. Lauraeus, M., Haltia, T., Saraste, M., and Wikström, M. (1991) Eur. J. Biochem. 197, 699-705[Medline] [Order article via Infotrieve]
8. Lauraeus, M., and Wikström, M. (1992) Biochemistry 31, 10054-10060[CrossRef][Medline] [Order article via Infotrieve]
9. Lemma, E., Schägger, H., and Kröger, A. (1993) Arch. Microbiol. 159, 574-578[CrossRef][Medline] [Order article via Infotrieve]
10. Powers, L., Lauraeus, M., Reddy, K. S., Chance, B., and Wikström, M. (1994) Biochim. Biophys. Acta 1183, 504-512[Medline] [Order article via Infotrieve]
11. Lauraeus, M., and Wikström, M. (1993) J. Biol. Chem. 268, 11470-11473[Abstract/Free Full Text]
12. Villani, G., Tattoli, M., Capitanio, N., Glaser, P., Papa, S., and Danchin, A. (1995) Biochim. Biophys. Acta 1232, 67-74[Medline] [Order article via Infotrieve]
13. Santana, M., Kunst, F., Hullo, M. F., Rapoport, G., Danchin, A., and Glaser, P. (1992) J. Biol. Chem. 267, 10225-10231[Abstract/Free Full Text]
14. Lemma, E., Simon, J., Schägger, H., and Kröger, A. (1995) Arch. Microbiol. 163, 432-438[Medline] [Order article via Infotrieve]
15. Saraste, M., Metso, T., Nakari, T., Jalli, T., Lauraeus, M., and van der Oost, J. (1991) Eur. J. Biochem. 195, 517-525[Medline] [Order article via Infotrieve]
16. von Wachenfeldt, C., and Hederstedt, L. (1992) FEMS Microbiol. Lett. 100, 91-100[CrossRef]
17. Bertsova, Y. V., Bogachev, A. V., and Skulachev, V. P. (1997) FEBS Lett. 414, 369-372[CrossRef][Medline] [Order article via Infotrieve]
18. Puustinen, A., Finel, M., Haltia, T., Gennis, R. B., and Wikström, M. (1991) Biochemistry 30, 3936-3942[CrossRef][Medline] [Order article via Infotrieve]
19. Winstedt, L., Yoshida, K., Fujita, Y., and von Wachenfeldt, C. (1998) J. Bacteriol. 180, 6571-6580[Abstract/Free Full Text]
20. Kunst, F., Ogasawara, N., Moszer, I., Albertini, A. M., Alloni, G., Asevedo, V., Bertero, M. G., Bessieres, P., Bolotin, A., Borchert, S., et al.. (1997) Nature 390, 249-256[CrossRef][Medline] [Order article via Infotrieve]
21. Sakamoto, J., Matsumoto, A., Oobuchi, K., and Sone, N. (1996) FEMS Microbiol. Lett. 143, 151-158[CrossRef][Medline] [Order article via Infotrieve]
22. Gilmour, R., and Krulwich, T. A. (1997) J. Bacteriol. 179, 863-870[Abstract/Free Full Text]
23. de Vrij, W., van den Burg, B., and Konings, W. N. (1987) Eur. J. Biochem. 166, 589-595[Medline] [Order article via Infotrieve]
24. Svensson, B., Lübben, M., and Hederstedt, L. (1993) Mol. Microbiol. 10, 193-201[CrossRef][Medline] [Order article via Infotrieve]
25. Puustinen, A., and Wikström, M. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 6122-6126[Abstract/Free Full Text]
26. Lemma, E., Hägerhäll, C., Geisler, V., Brandt, U., von Jagow, G., and Kröger, A. (1991) Biochim. Biophys. Acta 1059, 281-285[Medline] [Order article via Infotrieve]
27. Lemma, E., Unden, G., and Kröger, A. (1990) Arch. Microbiol. 155, 62-67[CrossRef][Medline] [Order article via Infotrieve]
28. von Wachenfeldt, C., and Winstedt, L. (1998) EBEC Short Rep. 10, 103
29. Johansson, P., and Hederstedt, L. (1999) Microbiology 145, 529-538[CrossRef][Medline] [Order article via Infotrieve]
30. van der Oost, J., von Wachenfeldt, C., Hederstedt, L., and Saraste, M. (1991) Mol. Microbiol. 5, 2063-2072[CrossRef][Medline] [Order article via Infotrieve]
31. Fridén, H., Hederstedt, L., and Rutberg, L. (1987) FEMS Microbiol. Lett. 41, 203-206[CrossRef]
32. Guérout-Fleury, A.-M., Shazand, K., Frandsen, N., and Stragier, P. (1995) Gene (Amst.) 167, 335-336[CrossRef][Medline] [Order article via Infotrieve]
33. Hoch, J. A. (1991) Methods Enzymol. 204, 305-320[Medline] [Order article via Infotrieve]
34. Hederstedt, L. (1986) Methods Enzymol. 126, 399-414[Medline] [Order article via Infotrieve]
35. Jünemann, S., Wrigglesworth, J. M., and Rich, P. R. (1997) Biochemistry 36, 9323-9331[CrossRef][Medline] [Order article via Infotrieve]
36. Meunier, B., Madgwick, S. A., Reil, E., Oettmeier, W., and Rich, P. R. (1995) Biochemistry 34, 1076-1083[CrossRef][Medline] [Order article via Infotrieve]
37. Jünemann, S., and Wrigglesworth, J. M. (1995) J. Biol. Chem. 270, 16213-16220[Abstract/Free Full Text]
38. Kita, K., Konishi, K., and Anraku, Y. (1984) J. Biol. Chem. 259, 3375-3381[Abstract/Free Full Text]
39. Leung, D., van der Oost, J., Kelly, M., Saraste, M., Hill, S., and Poole, R. K. (1994) FEMS Microbiol. Lett. 119, 351-358[CrossRef][Medline] [Order article via Infotrieve]
40. Lorence, R. M., Koland, J. G., and Gennis, R. B. (1986) Biochemistry 25, 2314-2321[CrossRef][Medline] [Order article via Infotrieve]
41. Borisov, V., Arutyunyan, A. M., Osborne, J. P., Gennis, R., and Konstantinov, A. A. (1999) Biochemistry 38, 740-750[CrossRef][Medline] [Order article via Infotrieve]
42. Wood, P. M. (1984) Biochim. Biophys. Acta 768, 293-317[Medline] [Order article via Infotrieve]
43. Kelly, M. J. S., Poole, R. K., Yates, M. G., and Kennedy, C. (1990) J. Bacteriol. 172, 6010-6019[Abstract/Free Full Text]
44. Kita, K., Konishi, K., and Anraku, Y. (1984) J. Biol. Chem. 259, 3368-3374[Abstract/Free Full Text]
45. Miyoshi-Akiyama, T., Hayashi, M., and Unemoto, T. (1993) Biochim. Biophys. Acta 1141, 283-287[Medline] [Order article via Infotrieve]
46. Sakamoto, J., Handa, Y., and Sone, N. (1997) J. Biochem. (Tokyo) 122, 764-771[Abstract/Free Full Text]
47. Garcia-Horsman, J. A., Berry, E., Shapleigh, J. P., Alben, J. O., and Gennis, R. B. (1994) Biochemistry 33, 3113-3119[CrossRef][Medline] [Order article via Infotrieve]
48. Gray, K. A., Grooms, M., Myllykallio, H., Moomaw, C., Slaughter, C., and Daldal, F. (1994) Biochemistry 33, 3120-3127[CrossRef][Medline] [Order article via Infotrieve]
49. Raitio, M., and Wikström, M. (1994) Biochim. Biophys. Acta 1186, 100-106[CrossRef]
50. Tamegai, H., and Fukumori, Y. (1994) FEBS Lett. 347, 22-26[CrossRef][Medline] [Order article via Infotrieve]
51. Poole, R. K. (1994) Antonie Leeuwenhoek 65, 289-310[CrossRef][Medline] [Order article via Infotrieve]
52. Jünemann, S. (1997) Biochim. Biophys. Acta 1321, 107-127[Medline] [Order article via Infotrieve]
53. Jünemann, S., Rich, P. R., and Wrigglesworth, J. M. (1995) Biochem. Soc. Trans. 23, 157S[Medline] [Order article via Infotrieve]
54. Hill, B. C., Hill, J. J., and Gennis, R. B. (1994) Biochemistry 33, 15110-15115[CrossRef][Medline] [Order article via Infotrieve]
55. Muntyan, M. S., Ludwig, B., Zickermann, I., and Starshinova, N. P. (1998) FEBS Lett. 429, 216-220[CrossRef][Medline] [Order article via Infotrieve]
56. Mitchell, R., Moody, A. J., and Rich, P. R. (1995) Biochemistry 34, 7576-7585[CrossRef][Medline] [Order article via Infotrieve]
57. Krasnoselskaya, I., Arutjunjan, A. M., Smirnova, I., Gennis, R., and Konstantinov, A. A. (1993) FEBS Lett. 327, 279-283[CrossRef][Medline] [Order article via Infotrieve]
58. Tsubaki, M., Hori, H., Mogi, T., and Anraku, Y. (1995) J. Biol. Chem. 270, 28565-28569[Abstract/Free Full Text]
59. Sun, J., Osborne, J. P., Kahlow, M. A., Kaysser, T. M., Gennis, R. B., and Loehr, T. M. (1995) Biochemistry 34, 12144-12151[CrossRef][Medline] [Order article via Infotrieve]
60. Moody, A. J., Rumbley, J. N., Gennis, R. B., Ingledew, W. J., and Rich, P. R. (1993) Biochim. Biophys. Acta 1141, 321-329[Medline] [Order article via Infotrieve]
61. Morgan, J. E., Verkhovsky, M. I., Puustinen, A. E., and Wikström, M. (1993) Biochemistry 32, 11413-11418[CrossRef][Medline] [Order article via Infotrieve]
62. Gilmour, R., and Krulwich, T. A. (1996) Biochim. Biophys. Acta 1276, 57-63[Medline] [Order article via Infotrieve]
63. Grinkevich, V. A., Lysenko, A. M., Muntyan, M. S., Skripnikova, E. V., and Afrikyan, E. K. (1997) Biokhimiya 62, 718-724
64. Muntyan, M. S., Ustiyan, V. S., Viryasov, M. B., and Skulachev, V. P. (1995) Biochem. Biophys. Res. Commun. 207, 55-61[CrossRef][Medline] [Order article via Infotrieve]
65. Azarkina, N. V. (1995) Biokhimiya 60, 211-216
66. Simpson, H., Denis, M., and Malatesta, F. (1997) Biosci. Rep. 17, 343-346[CrossRef][Medline] [Order article via Infotrieve]
67. Cunningham, L., Pitt, M., and Williams, H. D. (1997) Mol. Microbiol. 24, 579-591[CrossRef][Medline] [Order article via Infotrieve]
68. Barquera, B., Toledo, M., Finel, M., Wikström, M., and Garcia-Horsman, J.-A. (1996) EBEC Short Rep. 9, 79
69. Matsushita, K., Ebisuya, H., and Adachi, O. (1992) J. Biol. Chem. 267, 24748-24753[Abstract/Free Full Text]
70. Peschek, G. A., Alge, D., Fromwald, S., and Mayer, B. (1995) J. Biol. Chem. 270, 27937-27941[Abstract/Free Full Text]
71. Puustinen, A., Morgan, J. E., Verkhovsky, M., Thomas, J. W., Gennis, R. B., and Wikström, M. (1992) Biochemistry 31, 10363-10369[CrossRef][Medline] [Order article via Infotrieve]
72. Sone, N., and Fujiwara, Y. (1991) FEBS Lett. 288, 154-158[CrossRef][Medline] [Order article via Infotrieve]
73. Zickermann, I., Tautu, O. S., Link, T. A., Korn, M., Ludwig, B., and Richter, O.-M. H. (1997) Eur. J. Biochem. 246, 618-624[Medline] [Order article via Infotrieve]
74. Poole, R. K. (1988) in Bacterial Energy Transduction (Anthony, C., ed) , pp. 231-291, Academic Press, London

Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.
Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 J BiochemHome page
T. Mogi
Effects of Replacement of Low-Spin Haem b by Haem O on Escherichia coli Cytochromes bo and bd Quinol Oxidases
J. Biochem., May 1, 2009; 145(5): 599 - 607.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
M. Contreras-Zentella, G. Mendoza, J. Membrillo-Hernandez, and J. E. Escamilla
A Novel Double Heme Substitution Produces a Functional bo3 Variant of the Quinol Oxidase aa3 of Bacillus cereus: PURIFICATION AND PARTIAL CHARACTERIZATION
J. Biol. Chem., August 22, 2003; 278(34): 31473 - 31478.
[Abstract] [Full Text] [PDF]

Home page
 J. Bacteriol.Home page
B. D. Kana, E. A. Weinstein, D. Avarbock, S. S. Dawes, H. Rubin, and V. Mizrahi
Characterization of the cydAB-Encoded Cytochrome bd Oxidase from Mycobacterium smegmatis
J. Bacteriol., December 15, 2001; 183(24): 7076 - 7086.
[Abstract] [Full Text] [PDF]

Home page
 J. Bacteriol.Home page
L. Winstedt and C. von Wachenfeldt
Terminal Oxidases of Bacillus subtilis Strain 168: One Quinol Oxidase, Cytochrome aa3 or Cytochrome bd, Is Required for Aerobic Growth
J. Bacteriol., December 1, 2000; 182(23): 6557 - 6564.
[Abstract] [Full Text]

Home page
 J. Biol. Chem.Home page
V. B. Borisov, S. E. Sedelnikova, R. K. Poole, and A. A. Konstantinov
Interaction of Cytochrome bd with Carbon Monoxide at Low and Room Temperatures. EVIDENCE THAT ONLY A SMALL FRACTION OF HEME b595 REACTS WITH CO
J. Biol. Chem., June 15, 2001; 276(25): 22095 - 22099.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Azarkina, N.
Right arrow Articles by Konstantinov, A. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Azarkina, N.
Right arrow Articles by Konstantinov, A. A.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 1999 by the American Society for Biochemistry and Molecular Biology.
Advertisement
spacer
Advertisement
Advertisement