From the Institut für Biochemie,
Medizinische Universität zu Lübeck, Ratzeburger Allee 160, 23538 Lübeck, Germany and the § Forschungszentrum
Borstel, Zentrum für Medizin und Biowissenschaften,
Parkallee 1-40, 23845 Borstel, Germany
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In this study we re-examined the inducible
cytochrome b558/566 from the archaeon
Sulfolobus acidocaldarius (DSM 639), formerly thought to be
a component of a terminal oxidase (Becker, M., and Schäfer, G. (1991) FEBS Lett. 291, 331-335). An improved purification method increased the yield of the protein and allowed more detailed investigations. Its molecular mass and heme content have been found to
be 64,210 Da and 1 mol of heme/mol of protein, respectively. It is only
detectable in cells grown at low oxygen tensions. The composition of
the growth medium also exerts significant influence on the cytochrome
b558/566 content of S. acidocaldarius membranes. The cytochrome exhibits an extremely
high redox potential of +400 mV and shows no CO reactivity; a ligation
other than a His/His-coordination of axial ligands appears likely. It
turned out to be highly glycosylated (more than 20% of its molecular
mass are sugar residues) and is probably exposed to the outer surface
of the plasma membrane. The sugar moiety consists of several
O-glycosidically linked mannoses and at least one
N-glycosidically linked hexasaccharide comprising two
glucoses, two mannoses, and two N-acetyl-glucosamines. The gene of the cytochrome (cbsA) has been sequenced, revealing
an interesting predicted secondary structure with two putative
-helical membrane anchors flanking the majority of a mainly
-pleated sheet structure containing unusually high amounts of serine
and threonine. A second gene (cbsB) was found to be
cotranscribed. The latter displays extreme hydrophobicity and is
thought to form a functional unit with cytochrome
b558/566 in vivo, although it did
not copurify with the latter. Sequence comparisons show no similarity
to any entry in data banks indicating that this cytochrome is indeed a
novel kind of b-type hemoprotein. A cytochrome
c analogous function in the pseudoperiplasmic space of
S. acidocaldarius is discussed.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Sulfolobus acidocaldarius has been described as an obligate aerobic, facultatively chemolithoautotrophic, hyperthermoacidophilic archaeon growing at 75-80 °C and pH 2-2.5 (1). However, it grows much faster under heterotrophic conditions. Its respiratory chain consists of at least one succinate dehydrogenase (2, 3), two Rieske proteins (4), and two terminal oxidases (5, 6). In S. acidocaldarius membranes, three different a-type and two different b-type but no c-type cytochromes can be detected. Most cytochromes can be attributed to either one of the two oxidase complexes. Cytochrome a587 (SoxC) and cytochrome aa3 (SoxB) are part of the SoxABCD quinol oxidase (7, 8), whereas a second cytochrome a587 (SoxG) and a cytochrome b562 (SoxM) are constituents of the SoxM terminal oxidase complex (9). However, a second b-type cytochrome cannot be attributed to any of these complexes. Previously (10), cytochrome b558/566, named after its absorption maxima in the redox difference spectrum, was thought to participate in terminal electron transport due to its positive redox potential of >300 mV, its putative heme and copper content, and the CO difference spectrum typical for an o-type cytochrome obtained with partially purified membrane solubilisates. Because the function of this membrane-residing cytochrome remained obscure and could not be attributed to redox systems of respiratory electron transport, a genetic approach toward its further characterization was taken.
In this report, we present an improved purification method providing high yields of this unusual cytochrome for a fundamentally revised spectroscopic and protein-chemical characterization. Cytochrome b558/566 is shown to be highly glycosylated, and the first results of its glycosylation pattern are reported. In addition, an operon has been identified and sequenced encoding this interesting and novel hemoprotein; the transcription pattern is described. From the primary sequence, we concluded that in contrast to previous speculations, this cytochrome has no function as part of a terminal oxidase. A function as an ectoenzyme participating in periplasmic metabolism is discussed on the basis of its significant up- and down-regulation by modifying the growth conditions of the Sulfolobus cells.
![]() |
EXPERIMENTAL PROCEDURES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Bacterial Strains, Plasmids, and Culture Conditions
S. acidocaldarius cells (DSM 639) were grown heterotrophically in a mineral salt medium at 78 °C and at pH 2.5 in a 50-liter fermenter as described previously (11). To establish oxygen-limiting conditions throughout cell growth, aeration was started with a 12 liter/h airflow and was increased in a step-wise manner up to 35 liters/h in parallel to the increasing cell density.
Escherichia coli XL2-blue (Stratagene) was grown at
37 °C in LB medium (12) supplemented with 50 µg of ampicillin/ml.
The plasmid pBluescript II SK was purchased from
Stratagene (Heidelberg, Germany), and pSP64 was purchased from
Serva (Heidelberg, Germany).
Cell Harvest and Membrane Preparation
S. acidocaldarius cells were harvested in the late
logarithmic phase at an optical density of 1.4-1.5 by continuous flow
separation using a Westfalia separator. The cells were resuspended in
50 mM Mes,1 1 mM EDTA, pH 5.5; centrifuged at 7800 × g;
resuspended in the same buffer; frozen in liquid nitrogen; and stored
at 70 °C. Typical yields were 2.4-3 g of wet cell material per
liter of culture medium. Cells were disrupted in a Manton-Gaulin press, and membranes were prepared as described previously (11).
Isolation of Cytochrome b558/566
Hydrophobic Interaction Chromatography-- For chaotropic preextraction, the membranes were diluted to a protein concentration of 10 mg/ml in 50 mM potassium phosphate, 30 mM sodium pyrophosphate, pH 7.5. After stirring for 1 h at room temperature, the suspension was centrifuged (120,000 × g for 1 h at 4 °C). The membrane pellets were resuspended in 50 mM Tris/HCl, pH 7.3, and AS was added to a final concentration of 500 mM. DM was added to a final concentration of 20 mM. After stirring for 1 h at room temperature, the solution was centrifuged (as above) to remove insoluble components. The following steps were performed at 4 °C. A saturated solution of AS was slowly added to the supernatant to achieve 50% saturation. This solution was applied to a propyl-agarose column (Sigma P-5268; 1.5 cm in diameter, 16 cm in length) equilibrated with 50% saturated AS, 1 mM DM, 25 mM Tris/HCl, pH 7.3, at 0.5 ml/min. Subsequently, the column was washed with 120 ml of the same buffer. Elution was performed by a step-wise decrease in AS concentration and by a change in detergent (step 1: 40% AS saturation, 0.2 mM DM, 150 ml; step 2: 40% AS saturation, 0.5% SB-12, 120 ml; step 3: 20% AS saturation, 0.2 mM DM, 120 ml; all solutions with 25 mM Tris/HCl, pH 7.3). A minor fraction of the cytochrome b558/566 eluted at step 1 with large amounts of contaminating proteins. The major part eluted at step 2 in a much purer form. Fractions from step 2 were collected and concentrated by ultrafiltration on a PM-30 membrane (Amicon, Beverly, MA) to 3-5 ml.
Gel Filtration-- The concentrated cytochrome fraction was further purified by gel filtration (50 mM Tris/HCl, 0.5 mM DM; flow, 1 ml/min; Highload Superdex 200, Amersham Pharmacia Biotech). Elution was monitored by the absorbance at 280 nm and 430 nm. Fractions containing the cytochrome b558/566 were pooled and concentrated by ultrafiltration (as above). Further contaminating proteins were removed by digestion with trypsin (about 1 mg of trypsin/10 mg of protein) and another gel filtration step (as above). If the cytochrome preparations were to be used for sugar analyses other than the chemical deglycosylation procedure (see below), both gel filtrations were carried out with 0.5% SB-12 instead of DM.
Protein and Heme b Determination
Protein concentrations were determined by the modified Lowry
method in the presence of detergents (13) using bovine serum albumin as
standard or with the Bio-Rad DC protein assay (Bio-Rad). Heme b concentrations were determined as pyridine
hemochromogen according to standard methods (14), using a molar
extinction coefficient of = 22,100 M
1cm
1.
Spectroscopic Methods
Absorption spectra were recorded at room temperature using a HP 8453 diode array spectrophotometer or at liquid nitrogen temperature using a SIGMA ZWS-II dual wavelength spectrophotometer equipped with a laboratory-designed low temperature device as described in Ref. 15. Spectro-electrochemical determination of the redox potential was performed essentially according to Ref. 16 using a laboratory-designed multireflection titration cell in conjugation with the quartz fiber optics of a DW-2 spectrophotometer (15).
Electron paramagnetic resonance spectra were recorded with an X-band Bruker ER 200 D-SRC spectrometer equipped with an ESR 910 continuous flow helium cryostat from Oxford instruments. Copper content was determined using a Hitachi 180-80 Zeeman atomic absorption spectrophotometer set at 324.8 nm. For the measurement of the the iron content, the spectrophotometer was set at 248.3 nm.
Gas Liquid Chromatography and Mass Spectrometry (GLC-MS and MALDI-Time of Flight MS)
GLC was performed on a Varian 3700 chromatograph (Varian)
equipped with a fused-silica gel SPB-5 column using a temperature gradient of 150 °C (3 min) 320 °C at 5 °C/min. GLC-MS was
performed on a HP 5989A instrument (Hewlett-Packard) equipped with an
HP-5 column under the same chromatographic conditions as for GLC.
MALDI-MS was performed with a Bruker-Reflex II (Bruker-Franzen, Bremen, Germany) instrument in the positive linear time of flight mode at an acceleration voltage of 28.5 kV. Samples were dispersed in TA (0.1% CF3COOH-acetonitrile, 2:1 by volume) in a concentration of approximately 1 µg/ml. Part of this mixture (1 µl) was added to 15 µl of matrix solution (saturated sinapic acid (Aldrich) in TA). Aliquots of 0.5 µl were deposited on a metallic sample holder and analyzed immediately after drying in a stream of air.
Gel Electrophoreses and Western Blots
Polyacrylamide gel electrophoresis (PAGE) was carried out in the presence of SDS (17) using a 15% acrylamide gel with 1% bisacrylamide as cross-linker. Cyanogen bromide-cleaved fragments (see below) of the cytochrome b558/566 were separated by electrophoresis according to the method described by Schägger and von Jagow (18) using a 16.5% separating gel, a 10% spacer gel, and a 4% stacking gel, all with 3% bisacrylamide as cross-linker. Proteins were visualized by staining with Coomassie Brilliant Blue R-250. Glycoprotein staining was performed as described in Ref. 19.
Polyacrylamide gels were blotted on polyvinylidene difluoride membranes at 40 mA for 1-2 h with an electroblot apparatus (Semi-Phor, Hoefer Scientific Instruments) using 50 mM sodium borate, 0.02% SDS, 10% methanol, pH 9.0, as a transfer buffer.
Isoelectric focusing was carried out using 8% acrylamide gels with 3% cross-linker containing 4% ampholytes and 17% glycerol. Ampholytes (Servalyt AG 3-10, Servalyt AG 4-6, and Servalyt T 2-4) were purchased from Serva.
Deglycosylation and Sugar Analysis
Chemical deglycosylation was performed using a commercially available kit (K-500, Oxford GlycoSystems, Abingdon, United Kingdom) that cleaves both O- and N-linked sugar residues by the use of trifluoromethane sulfonic acid.
For total sugar analysis, purified cytochrome b558/566 (1 mg) was hydrolysed in 1 ml of 2 M CF3COOH (100 °C for 4 h), reduced (NaBH4), per-O-acetylated (acetanhydride in pyridine, 1:1.5, v/v, 85 °C for 20 min), and analyzed by GLC and GLC-MS (20).
O-linked sugars were liberated by -elimination from
cytochrome b558/566 (1 mg) with 2 M
NaBH4 in 0.1 M NaOH (45 °C for 16 h)
(21). The mixture was neutralized, per-O-acetylated, and analyzed by GLC-MS.
A third aliquot (1 mg) was used to determine N-linked sugars
by heating with hydrazine (1 ml) in a sealed tube (100 °C for 12 h). After the reagent was evaporated under reduced pressure, sugars were N-acetylated (1 ml of saturated aqueous
NaHCO3 and 15 µl of acetic acid anhydride for 30 min).
The N-acetylation was repeated twice. After neutralization
with ion exchanger (IR 120, H+ form), the crude
oligosaccharide preparation was purified using a gel permeation
chromatography column (2 × 112 cm, Sephadex G-10) in water
monitoring the eluate at 206 nm (Uvicord, Amersham Pharmacia Biotech).
Oligosaccharide fractions were combined and lyophilized. One aliquot
() of the oligosaccharide was analyzed for its components by
GLC-MS analysis described above.
Chemical Cleavage and Amino Acid Sequencing
Purified samples of either intact cytochrome
b558/566 or of deglycosylated cytochrome (about
20-50 µg) were cleaved by 0.75 M cyanogen bromide in
75% formic acid in a final volume of 400 µl. The reaction mixture
was incubated for 1 h at 37 °C, diluted by addition of 400 µl
of distilled water, frozen at 70 °C, and dried in a Speed Vac
concentrator. The dried samples were resuspended in distilled water,
and the evaporation was repeated. Finally, samples were resuspended in
20 µl of water.
The fragments were separated by SDS-PAGE, blotted on polyvinylidene difluoride membranes, and stained with Coomassie Brilliant Blue R-250. Bands were cut out, and the amino acid sequence was determined by automated microscale Edman degradation.
DNA Techniques
All general cloning procedures were performed according to standard methods (12). Genomic DNA of S. acidocaldarius was prepared according to the method of Marmur (22), and plasmid DNA was isolated from E. coli cells on a small scale by lysis with lysozym and Triton X-100 (23) or on a large scale by anion exchange chromatography (Jetstar, Genomed, Bad Oeynhausen, Germany).
All nonradioactive labeling, blotting, hybridization, and chemiluminescence detection techniques were performed as described in detail in Ref. 24.
Cloning Procedure
The longest amino acid sequence obtained (MYEVDTAGIYYTPVAA) was chosen to derive two oligonucleotide probes (probe 1, 5'-CCWGCWGT-RTCWACYTCRTACAT-3'; probe 2, 5'-ATATAYTAYACWCCWGTWGC-WGC-3'). The oligonucleotides were 3'-end-labeled with digoxygenin-11-ddUTP (24).
S. acidocaldarius genomic DNA was digested with various
restriction enzymes, separated on agarose gels, and blotted by vacuum onto nylon membrane (Hybond-N, Amersham Pharmacia Biotech). A 2.8-kb
PstI/XbaI fragment positive with probe 1 was
chosen for insertion into pSP64 and cloning in E. coli
XL2-blue (plasmid pCBS1). Transformation into E. coli cells
was performed as described in Ref. 25. A 0.9-kb SspI
fragment out of the larger 2.8 kb fragment also positive with probe 1 was subcloned into pBluescript II SK (plasmid pCBS2).
Sequencing of the fragments was performed nonradioactively with Thermo
Sequenase kit (Amersham Pharmacia Biotech) using a GATC 1500 direct
blotting electrophoresis device (GATC) and the DIG system from
Boehringer Mannheim as described in Ref. 26.
Isolation of RNA and Northern Blot Hybridization
Total RNA was extracted from S. acidocaldarius cells with guanidium isothiocyanate and phenol using a commercially available kit (Roti-Quick-Kit, Carl Roth, Karlsruhe, Germany). Northern blotting and hybridization was performed as in Ref. 24.
Two RNA probes were produced by in vitro transcription of linearized DNA template (plasmid pCBS2 cut with XbaI or with BglII) with T7 polymerase and the DIG RNA labeling kit (Boehringer Mannheim) as described in Refs. 27 and 24. Hybridization and chemiluminescence detection was also performed as in Ref. 27.
Influence of Growth Medium on Cytochrome b558/566 Content of Membranes
To examine the influence of different growth media, S. acidocaldarius cells were grown on a small scale in a 1-liter fermenter under oxygen-limiting conditions. Select yeast extract was purchased from Life Technologies, Inc.; neutralized bacteriological peptone was from Oxoid (Basingstoke, United Kingdom); casein (enzymatic hydrolysate), L-leucine, and L-isoleucine were from Sigma; and 2-oxoglutaric acid was from Fluka (Deisenhofen, Germany).
Cells were harvested by centrifugation at 7800 × g.
Cells were disrupted for 5 min by ultrasonification, and membranes were prepared by down-scaling the procedure described previously (11). The
cytochrome b558/566 content of the membranes was
calculated based on the ascorbate-reduced minus ferricyanide-oxidized
difference spectrum using an extinction coefficient of
566-590 = 11,200 M
1cm
1, determined by pyridine
hemochromogen. The protein concentration of the membranes was
determined as described above.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Preparation of Cytochrome b558/566-- The cytochrome composition of the membranes changes according to the oxygen level at which the cells are grown (7). Under low oxygenation, the expression of cytochrome b558/566 strongly increases, whereas high oxygen levels (maximum air flow) suppress the production of this cytochrome nearly completely (15).
To isolate the protein, purified membranes were subjected to chaotropic pre-extraction with sodium pyrophosphate, which removed loosely bound proteins from the membrane. DM was used for solubilization as this detergent does not induce spectral changes of a- or b-type cytochromes (7). High ionic strength, achieved by addition of 500 mM AS during the solubilization step, improved the efficiency of the subsequent chromatographic preparation steps. The following hydrophobic interaction chromatography and gel filtration (see "Experimental Procedures") separates the cytochrome b558/566 from other cytochromes. Fig. 1 documents the process of purification resulting in a single protein band (right lane), susceptible to Coomassie Blue staining but not to silver staining. This unusual behavior led us to suspect that cytochrome b558/566 might be a glycoprotein. Staining for sugar residues (not shown) confirmed this assumption. It was further corroborated by the observation that alkali treatment caused a considerable decrease of apparent molecular mass (not shown). This unexpected result also explains the observation that cytochrome b558/566 is resistant to protease degradation (trypsin or V8; data not shown), a feature that was then included in the purification procedure. A tryptic digestion cleaved remaining contaminating proteins, and the final gel filtration removed these cleaved proteins as well as the added trypsin (Fig. 1, lane 5). The use of SB-12 instead of DM did not induce any spectral changes of cytochrome b558/566 but led to a minor decrease in long-term stability and a slight increase in susceptibility to proteolytic digestion.
|
Spectral Characterization--
In native membranes, cytochrome
b558/566 is the only cytochrome present in
significant amounts that is readily reducible by ascorbate (6, 15).
Difference spectra (reduced minus oxidized) of the purified cytochrome
b558/566 resembled the spectral features of
ascorbate-reduced S. acidocaldarius membranes, exhibiting
maxima at 566 and 558 nm and a minimum at 561 nm in the -band region and lower maxima at 530 and 538 nm in the
-region of the visible spectrum (Fig. 2). The Soret band in the
reduced minus oxidized difference spectrum was located at 430 nm. In
the reduced spectrum it was found at 430 nm as well, whereas it shifted
to 419 nm in the oxidized spectrum. The differential extinction
coefficients are
566-575 = 10,800 M
1cm
1,
566-590 = 11,200, and
430-439 = 65,900 M
1cm
1 based on the heme
b content determined as pyridine hemochromogen. At liquid
nitrogen temperature, the
-bands clearly resolved into two distinct
and narrow peaks at 563 and 553 nm, whereas the
-bands moved to 527 and 534 nm (data not shown). The pyridine hemochrome spectrum (Fig. 2)
with a maximum at 556 nm indicates that the purified compound contained
only heme b. In the above spectral region, neither the
isolated cytochrome, prepared according to the described procedure, nor
native membranes displayed a CO-inducible difference spectrum in the
reduced state, thus indicating a hexa-coordinated heme center without
exchangeable ligand positions. This is in sharp contrast to former
observations, in which the cytochrome had been extracted from membranes
immersed in an imidazole buffer during detergent extraction (10).
Actually, prolonged exposure of membranes or detergent extracts to
imidazole (0.1-0.6 M) causes the appearance of a confluent
-peak at 560-562 nm (28) with a high absorption coefficient, a
behavior that we were able to reproduce with the purified cytochrome.
Under the latter conditions, a reduced minus oxidized difference
spectrum resulted, which upon addition of CO clearly indicated the
formation of a carbon monoxide compound yielding a sharp trough at 560 nm (556 nm at liquid nitrogen temperature) in the difference spectrum
(reduced plus CO, minus reduced) (Fig. 2).
|
|
Molecular Properties-- When using a pH 2-6 polyacrylamide gel, the pI was found to be in the range of 4-4.5 (data not shown). The apparent molecular mass (SDS-PAGE) was about 66 kDa (Fig. 1). This is in good agreement with the molecular mass determined by mass spectrometry: the positive ion MALDI-time of flight mass spectrum of cytochrome b558/566 recorded in the linear mode is shown in Fig. 4. The spectrum comprises two prominent mass peaks representing the single- and double-charged quasimolecular ions at m/z = 64,210 and 32,091, respectively.
|
|
Isolation and Sequencing of the Gene-- Direct N-terminal amino acid sequence analysis of purified cytochrome b558/566 after SDS-PAGE and electroblotting onto polyvinylidene difluoride membrane failed, irrespective whether or not the sample was deglycosylated. Only CNBr cleavage of a deglycosylated sample of cytochrome b558/566 produced a peptide fragment sufficient for synthesis of oligonucleotide probes, suitable for Southern blot hybridization. Restriction enzyme-digested chromosomal DNA from S. acidocaldarius gave positive signals only with probe 1. Probe 2 did not give any signal due to two protein sequencing errors, detected after the gene had been sequenced. The gene, including its flanking regions, was cloned and sequenced from a 2.8-kb PstI/XbaI-fragment and from a 0.9-kb SspI/SspI-fragment subclone as described under "Experimental Procedures."
Gene Organization and Transcription Analysis-- Fig. 6 demonstrates the organization of the cytochrome b558/566 operon. Three open reading frames could be found within the sequenced DNA region: an open reading frame from nucleotide 179 to 1564 encoding cbsA (cytochrome b558/566 from Sulfolobus), a second open reading frame from nucleotide 1564 to 2490 encoding cbsB, and a third open reading frame (orf1) starting at nucleotide 2568 and extending beyond the end of the sequenced clone at nucleotide 2769. Fig. 7 shows both the gene and the protein sequences of cytochrome b558/566 and of an extremely hydrophobic protein (CbsB) not identified by protein chemistry so far. Some characteristic features are emphasized, such as, for example, a putative Box A and a possible terminating structure following the cbsB gene; possible transmembrane sections are underlined, and the only three histidines that are candidates for heme b coordination are circled.
|
|
Amino Acid Sequence and Composition of Cytochrome b558/566 and CbsB-- The cbsA gene product contains 462 amino acids and has a predicted molecular mass of 50,736 Da. However, sequence comparisons with protein data bases revealed no significant similarity to any other cytochrome or known hemoprotein.
In accordance with the high degree of glycosylation of the protein (>20%), the amino acid composition displays a high content of serine and threonine (>9% each, Table 1 and Fig. 6). In addition, 16 amino acid triplet sequences of NXS or NXT denote possible N-glycosylation sites.
|
|
Influence of Growth Medium on Cytochrome b558/566 Content of Membranes-- Previous experiments showed that even under oxygen-limiting conditions, S. acidocaldarius membranes contain only barely detectable amounts of cytochrome b558/566 when the cells had been grown in a medium largely deprived of nitrogen sources, i.e. yeast extract and L-glutamate were omitted and ammonium sulfate was reduced from 10 to 1.5 mM.2 This was the basis for experiments to grow S. acidocaldarius in media containing different carbon and nitrogen sources. The standard medium contained sucrose (0.2%), ammonium sulfate (10 mM), glutamate (5.8 mM), and 0.1% yeast extract. The most significant effects were perceived by replacement of the yeast extract. Under oxygen-limiting conditions, when the yeast extract was substituted for by peptone (data not shown), the content of cytochrome b558/566 doubled; when it was substituted for by casein hydrolysate, the content more than tripled (Fig. 9, column 4). Because the main component of peptone and casein are enzymatically digested proteins, a few selected amino acids were tested as to whether they could produce the same effect when they replaced the yeast extract. A mixture of L-leucine and L-isoleucine (each 1.25 mM in the growth medium) also caused a doubling in cytochrome b558/566 content (data not shown), whereas L-isoleucine alone (2.5 mM) caused more than a tripling of the content (Fig. 9, column 5). The content of the other cytochromes did not change significantly under any of these conditions. When the yeast extract was replaced by L-tryptophan, no growth occurred.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The purification method by hydrophobic interaction chromatography previously described in Ref. 29 and further improved here led to a significant increase in the yield of cytochrome b558/566 preparations, unexpectedly revealing its character as a membrane-residing glycoprotein. Most likely due to its high glycosylation, the protein does not stain with silver, a property that in previous studies (10) caused the erroneous attribution of the spectrum to a well-staining 30-kDa band that in reality represented a copurifying contaminant polypeptide. The correct assignment to a 66-kDa Coomassie-staining band was now confirmed by mass spectrometry, which gave a single signal at 64.2 kDa for the molecular ion, thus simultaneously illustrating the homogeneity of the preparation. Consequently, the revised estimate of its heme content amounts to 1 heme b/mol, adjusting the actual values of 0.6-0.8 to the next integer stoichiometry. No tightly bound copper was associated with these preparations.
Structure and Localization-- Cytochrome b558/566 is an integral membrane protein and can only be solubilized by strong detergents. It is only the fourth glycoprotein found in the genus Sulfolobus. From S. acidocaldarius, the S-layer protein has been described as a glycoprotein (30). Thermopsin, an acidic protease partially associated with the cells, partially found in the medium, is thought to be a glycoprotein as well, because it contains 11 potential N-glycosylation sites (31). In S. shibatae, two flagellin proteins with identical N-terminal sequences have been found that stained positively for glycoproteins (32). To our knowledge, the composition and the structure of the sugar moiety has not been elucidated in these examples. Hence, this study is the first time that the sugar part of a glycoprotein from the genus Sulfolobus has undergone close examination.
Sequencing of the cbs operon provides interesting insights into the primary structure of this novel hemoprotein. The two hydrophobic stretches at the N and C termini (Fig. 8) are thought to serve as membrane anchors, whereas the hydrophilic major part of the protein is highly glycosylated and therefore is supposed to be exposed to the outer surface of the plasma membrane. Notably, secondary structure predictions for this middle part suggest mainly loop regions andProperties and Function--
An important alteration in the
purification procedure was to avoid imidazole in any buffer. Imidazole
presumably is able to alter the ligation of the heme-iron, replacing a
weaker ligand provided by the amino acid side chains of the protein. As
such, 9 out of 10 methionines located in the central hydrophilic core of the protein may be envisaged. Thus, imidazole treatment appears likely to pave the way for a further ligand exchange against CO. Cytochrome b558/566 prepared according to the
described protocol does not form a CO compound, but it was shown to
reversibly assume this property after exposure to 0.1 M
imidazole. Whether a bis-His or a His/Met ligation is present in the
cytochrome cannot be decided from the EPR spectra, because the only
unambiguously detected gz value falls into the overlap region
for both types of ligands. In analogy to c-type cytochromes
with His/Met ligation, the novel cytochrome
b558/566 exhibits a reduction potential >+350 mV, whereas species with bis-His ligation have reduction potentials in
the range of 0 to 400 mV (33). Furthermore, c-type
cytochromes with His/Met ligation distorted by, for example, imidazole
treatment also become capable of binding CO, whereas integral
c-type cytochromes are not (34). In this context, the
observed CO binding behavior of cytochrome
b558/566 can be sufficiently explained. But
also, even more unusual types of ligation cannot definitely be
excluded. For example, cytochrome f of the chloroplast
cytochrome b6f complex from
Brassica rapa binds heme with the N terminus of the
polypeptide chain as secondary ligand (35). However, the gz
value of 3.13 in the EPR spectrum of cytochrome
b558/566 is not in accord with this type of
ligation. Regardless how the heme group is coordinated, it is certainly
not embedded within the membrane-spanning regions of the protein,
because all three histidines providing at least the primary axial
ligand to the heme iron are also located in the large hydrophilic core
of the protein.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. Werner G. Purschke for general advice on DNA and RNA techniques and Walter Verheyen for skillful technical assistance. We thank Simone Kardinahl for performing the atomic absorption spectroscopy. We are indebted to Dr. Buko Lindner and Helga Lüthje for the MALDI-time of flight MS analysis.
![]() |
FOOTNOTES |
---|
* This work was supported by a grant from the Deutsche Forschungsgemeinschaft (Scha 125/17-3).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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) Y10108.
¶ To whom correspondence should be addressed. Tel.: 49-451-500-4060; Fax: 49-451-500-4068; E-mail: schaefer{at}physik.mu-luebeck.de.
1
The abbreviations used are: Mes,
2-(N-morpholino)ethanesulfonic acid; AS, ammonium sulfate;
DM, n-dodecyl--maltoside; MALDI, matrix-assisted laser
desorption/ionization; MS, mass spectrometry; SB-12,
N-dodecyl-N,N-dimethyl-ammonio-3-propane-sulfonate;
GLC, gas liquid chromatography; PAGE, polyacrylamide gel
electrophoresis; kb, kilobase(s).
2 Christian L. Schmidt, unpublished results.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|