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Originally published In Press as doi:10.1074/jbc.M302583200 on June 12, 2003

J. Biol. Chem., Vol. 278, Issue 34, 31473-31478, August 22, 2003
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A Novel Double Heme Substitution Produces a Functional bo3 Variant of the Quinol Oxidase aa3 of Bacillus cereus

PURIFICATION AND PARTIAL CHARACTERIZATION*

Martha Contreras-Zentella {ddagger}, Guillermo Mendoza §, Jorge Membrillo-Hernández ¶ and José Edgardo Escamilla {ddagger} ||

From the {ddagger}Instituto de Fisiología Celular, §Facultad de Medicina, and Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, Mexico City 04510, Mexico

Received for publication, March 13, 2003 , and in revised form, June 4, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
A novel bo3-type quinol oxidase was highly purified from Bacillus cereus PYM1, a spontaneous mutant unable to synthesize heme A and therefore spectroscopically detectable cytochromes aa3 and caa3. The purified enzyme contained 12.4 nmol of heme O and 11.5 nmol of heme B mg–1 protein. The enzyme was composed of two subunits with an Mr of 51,000 and 30,000, respectively. Both subunits were immunoreactive to antibodies raised against the B cereus aa3 oxidase. Moreover, amino-terminal sequence analysis of the 30-kDa subunit revealed that the first 19 residues were identical to those from the 30-kDa subunit of the B. cereus aa3 oxidase. The purified bo3 oxidase failed to oxidize ferrrocytochrome c (neither yeast nor horse) but oxidized tetrachlorohydroquinol with an apparent Km of 498 µM, a Vmax of 21 µmol of O2 min–1mg–1, and a calculated turnover of 55 s–1. The quinol oxidase activity with tetrachlorohydroquinol was inhibited by potassium cyanide and 2-n-heptyl 4-hydroxyquinoline-N-oxide with an I50 of 24 and 300 µM, respectively. Our results demonstrate that the bo3 oxidase of this mutant is not the product of a new operon but instead is a cytochrome aa3 apoprotein encoded by the qox operon of the aa3 oxidase of B. cereus wild type promiscuously assembled with hemes B and O replacing heme A, producing a novel bo3 cytochrome. This is the first reported example of an enzymatically active promiscuous oxidase resulting from the simultaneous substitution of its original hemes in the high and low spin sites.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Bacteria have exploited unique terminal oxidases depending on the natural habitats and modes of aerobic metabolism (1). In most cases, there is more than one terminal oxidase, so that the respiratory systems of bacteria are branched. According to the nature of the electron donor two types of terminal oxidases can readily be distinguished: the cytochrome c oxidases and the quinol oxidases (1).

Additionally, terminal oxidases contain different heme prosthetic groups that have provided a customary way of identification (i.e. aa3, bo3, caa3, bd, cbb3, ba3). However this classification is further complicated by the fact that under specific culture conditions (2–5) or as result of mutations (6–10), bacterial oxidases may be assembled promiscuously, accepting a different heme group to that present in its original structure. A single heme substitution (O replacing B) has been reported in the low spin heme site of cytochrome bo3 of over-expressing strains of Escherichia coli, resulting in the assembly of a functional cytochrome, oo3. It is noteworthy that the heme of the binuclear O2 reduction site was invariably heme O (6). Moreover, the substitution of heme O by heme B in the binuclear O2-reducing site in E. coli cyoE-deleted strains (cyoE encodes for farnesyl transferase that converts heme B to heme O) results in an inactive cytochrome bb3 enzyme (10). Similarly Zikermann et al. (7) reported that in Paracoccus denitrificans ctaB-deleted strains (the ctaB gene is an orthologue of cyoE), a bb3 variant of bo3 oxidase is enzymatically inactive. Examples of heme substitutions in the high spin heme site producing functional enzymes have been reported in several bacterial species; for example a novel b(o/a)3 cytochrome c oxidase was purified from Bacillus stearothermophilus K17 mutants defective in caa3-type oxidase (8). Likewise, the presence of a b(a/o)3-type oxidase was reported in Acetobacter aceti (5), a ca(a/o)3-type oxidase was found in Bacillus PS3 (4), and an a(a/o)3-type oxidase was isolated from cyanobacteria (11, 12). Either heme A or O can be bound to the O2-reducing site depending on the growth conditions; high O2 tension favors the assembly with heme A, whereas O2-limited conditions lead to a promiscuous substitution of heme A by heme O. This in good agreement with the fact that the synthesis of heme A from heme O requires an oxidizing/oxygenizing reaction of heme O (13, 14). Thus, O2 limitation would favor the accumulation of heme O.

Heme O is normally not present in the Bacillaceae family; however, the presence of heme O has been reported in wild type cells growing under limited aeration (3, 4) or in mutants deficient in the ctaA gene that encodes for the oxygenase responsible for the conversion of heme O into heme A (i.e. Bacillus subtilis RB829R (14)) as well as, in other genetically uncharacterized (presumably ctaA) mutants (B. subtilis FG83 (15) and Bacillus cereus PYM1 (16)). In none of these mutants were any type a cytochromes spectroscopically detected; instead, the presence of a functional type o oxidase in membranes was suggested based on a spectral studies of CO-binding and O2 displacement upon photodissociation at subzero temperatures (15, 17). The nature of these novel oxidases was not further explored.

Several promiscuous oxidases with single heme substitutions have been purified and characterized to varying extents (4, 6, 18). To our knowledge, an enzymatically active promiscuous oxidase bearing double heme substitution has yet to be described. In this work, we report on the purification and characterization of a novel cytochrome bo3 quinol oxidase from B. cereus PYM1. Our results indicate that the purified bo3 oxidase is a promiscuous enzyme that arises from the simultaneous insertion of hemes B and O in the low and high spin sites, respectively, of the original aa3 quinol oxidase apocytochrome. On the other hand, a functional promiscuous equivalent was not detected for the caa3 apocytochrome in the PYM1 strain, thus suggesting that double heme substitution producing active variants is a rare event among oxidases. The molecular and kinetic properties of the promiscuous bo3 oxidase were analyzed and compared with those of the original aa3 oxidase.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Bacterial Strains and Growth Conditions—A B. cereus wild type strain originally isolated by Andreoli et al. (19) and its spontaneous derivative, PYM1 (16), were used throughout this study. Bacterial cultures were grown at 30 °C in an 80-liter fermentor (Bioflo 5000, New Brunswick Scientific, Edison, NJ) containing 60 liters of nutrient sporulation medium phosphate (20) and stirred at 250 rpm with vigorous bubbling (60 liters of air min–1). Cells were harvested during sporulation at stage II (2 h into stationary phase), washed twice with 50 mM Tris-HCl, 5 mM CaCl2, and 5 mM MgSO4, pH 7.4, and resuspended (0.5 g of wet weight ml–1) in the same buffer.

Membrane Isolation and Protein Purification—Procedures for mechanical disruption of cells and membrane isolation were carried out as reported previously (16). For the present work, 0.1 mM phenylmethyl-sulfonyl fluoride and one tablet of protease inhibitors mixture (Roche Molecular Biochemicals) per 100 ml of cell suspension were included. The procedure for the purification of the bo3-like oxidase from PYM1 strain was similar to those used for the purification of the B. cereus oxidases aa3 and caa3 reported earlier, with minor modifications (21, 22). This procedure consisted of a bile salt extraction (sonication) of membrane particles followed by a 4% Triton X-100 solubilization of the resulting membrane residues and a 30% polyethylene glycol 6000 precipitation of the light brown-colored supernatant obtained after centrifugation of the Triton X-100 fraction. The purification was continued by two steps of anionic-exchange column chromatography in the following order: DEAE-Sepharose CL6B, QAE-Toyopearl (Sigma), and finally a HA-Ultrogel (Sigma) chromatography column. All purifications steps were carried out with 0.1% Triton X-100 added to buffers. Fractions displaying heme absorption (405 nm) were analyzed for oxidase activity using N,N,N',N'-tetramethyl-p-phenylendiamine (TMPD)1 or tetrachlorohydroquinol (TCHQ) reduced with ascorbate as electron donors. Those fractions containing activity were pooled and precipitated with 30% polyethylene glycol 6000 and resuspended in 5 mM Tris-HCl (potassium phosphate for HA Ultrogel column), pH 8.0, containing 10 µg ml–1 of azolectin. All purification was performed at 4 °C.

Spectroscopic Analysis—Conventional optical absorption difference spectra were recorded at room temperature in an SLM-Aminco DW 2000 spectrophotometer (SLM Instruments Inc.) using a 1-cm light path cuvettes. Samples were reduced with solid sodium dithionite, whereas references were oxidized with air (1 min of vortex agitation). The CO-cytochrome complexes were obtained by bubbling reduced preparations with CO during 5 min. An extinction coefficient of 22 mM–1 cm–1 for cytochrome b (555–575 nm) was used in the reduced minus oxidized spectra and of 160 mM–1 cm–1 (417–432 nm) for the cytochrome o CO adduct (17).

Photodissociation Spectroscopy—The purified preparation of cytochrome bo3-like (50 µg of protein) was lyophilized, then resuspended in aqueous 30% (v/v) ethylene glycol solution, and reduced by the addition of a few grains of dithionite in a 2-mm light path cuvette. The anoxic preparation was bubbled with CO for 5 min and then frozen in an ethanol-solid CO2 bath at 195 K for at least 5 min in the dark prior further cooling to 77 K in the sample compartment of the dual wavelength SLM-Aminco DW2000 spectrophotometer. The sample was scanned twice between 400 and 750 nm (using 500 nm as the reference wavelength), and the difference was plotted to obtain the base line. The frozen sample was then photolysed with three flashes of a photographic flash placed a few centimeters away from the window of the cuvette, and then the post-photolysis spectrum was obtained. The photodissociation difference spectrum of heme-CO compounds was obtained by subtracting the pre-photolysis spectrum from the post-photolysis spectrum (23, 24).

Electrophoretic and Western Blot Analyses—To decrease Triton X-100 content in purified preparations of cytochrome bo3-like, samples were passed through a 0.3 x 5-cm Bio-Beads SM-2 column (Bio-Rad) at 4 °C. SDS-PAGE was performed using a 10–16% gradient of acrylamide containing 2% SDS as described previously (16). Half of the gel was stained with Coomassie Blue (G-250, Sigma), and the other half was used for Western blot analyses with rabbit antiserum raised against B. cereus cytochrome aa3 as described previously (16)

Amino-terminal Sequence—Purified aa3- and bo3-like oxidases from wild type and PYM1 strains, respectively, were subjected to SDS-PAGE and electrophoretic transfer of proteins onto polyvinylidene difluoride membranes (25). Both proteins were sequenced by automated Edman degradation using a Beckman Spherogel Micro PTH (2 x 150 mm) column and a sequencer phase-gas (LF 3000, Beckman Instruments) equipped with a high-pressure liquid chromatography (HPLC) Gold system and a 168 photodiode array detector setting at 268 and 293 nm for signal and reference, respectively.

Respiratory Activities—Oxidase activity was determined polarographically at 30 °C in a YSI model 53 oxygen meter (Yellow Springs Instruments, Yellow Springs, OH) as described previously (16), using either 0.1 mM TMPD or 3.3 mM TCHQ reduced with 2.5 mM ascorbate.

Heme Analysis—Hemes were determined in a Waters chromatography system equipped with a Waters model 996 photodiode array detector and Waters Delta-Pak HPLC18 300° A (2 x 150 mm) reverse-phase HPLC column (MetaChem Technologies, Inc.). Hemes were eluted from the column by acetonitrile gradient in water containing 0.5% trifluoroacetic acid and detected by their absorbance at 405 nm as described previously (16). Heme preparations were extracted and purified from membranes or from the fractions obtained during each of the purification steps. Samples used for heme extraction were previously passed through a column of Bio-Beads.

Other Methods—Protein concentration was determined according to the method described by Lowry and modified by Markwell et al. (26) using bovine serum albumin as standard.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Purification of the bo3-like Oxidase from B. cereus PYM1— The preparation obtained after solubilization of membrane components by Triton X-100, was subjected to anionic-exchange chromatography in a DEAE-Sepharose CL6B column (15 x 7 cm). Two heme-containing fractions were separated, one of which was not adsorbed by the column and contained cytochromes b and c as judged by its reduced minus oxidized difference spectra (not shown). This fraction had a CO difference spectrum displaying typical absorbance bands for cytochrome type o (peak at 417 and trough at 427 nm). However, after HPLC analysis, the presence of heme O was not sustained, and only heme B was confirmed (data not shown). Moreover, this fraction did not present oxidase activity when either TMPD or TCHQ (reduced with ascorbate) were used as electron donors. By contrast, the equivalent chromatographic fraction from extracts of the wild type strain of B. cereus contained TMPD oxidase activity, and as reported earlier (22), further purification of this fraction confirmed the presence of cytochrome caa3 (not shown).

A second heme-containing fraction was eluted at a linear gradient of 175 mM NaCl (0–600 mM). This peak showed quinol oxidase activity with TCHQ. The reduced minus oxidized (peaks at 427, 555, and 562 nm) and CO difference spectra (peaks at 415, 538, and 573, troughs at 426 and 556 nm) of this fraction suggested the presence of b- and o-type cytochromes. After HPLC analysis we confirmed the presence of heme B (retention time of 27.5 min) and heme O (retention time of 34 min) in a 1:0.25 ratio (data not shown). Fractions containing TCHQ oxidase activity were pooled and precipitated with 30% polyethylene glycol 6000. The resuspended pellet was applied to a QAE-Toyopearl column. A fraction associated to cytochrome bo3-like was eluted at a linear gradient of 200 mM NaCl (0–600 mM). This peak contained TCHQ oxidase activity and the spectrally detectable cytochrome o CO adduct (data not shown). Fractions containing activity were precipitated as described above and applied to a HA-Ultrogel column to accomplish the purification to homogeneity of the cytochrome bo3-like of B. cereus PYM1. The enzyme was eluted at a linear gradient (0–500 mM)of65mM potassium phosphate buffer (Fig. 1A). The CO difference spectrum of the dithionite reduced preparation (Fig. 1B) and its HPLC analysis (Fig. 1C) confirmed the presence of cytochrome bo3-like in the purified preparation. A concentration of 11.5 and 12.4 nmol mg protein–1 for cytochromes b and o, respectively, was calculated from the dithionite reduced minus air oxidized difference and the CO difference spectra, respectively (Tables I and II). These results strongly suggest a mol-to-mol ratio between cytochromes b and o in the bo3-like oxidase of B. cereus PYM1. The results of the purification are summarized in Table I. The chromatographic behavior described for cytochrome bo3-like of the PYM1 strain was similar to that described previously for a cytochrome aa3 of the wild type strain (21, 22).



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  		FIG. 1.
Purification of the bo3 cytochrome oxidase from B. cereus PYM1. A, liquid chromatography in HA Ultrogel. The column was developed with a 5–500 mM potassium phosphate gradient, pH 8.0, and the peak of bo3 cytochrome oxidase was eluted at 65 mM. B, CO differential spectrum of the dithionite reduced of the pure enzyme. C, reverse-phase HPLC of hemes extracted with acetone-HCl from the pure enzyme. Hemes were eluted from the column by an acetonitrile gradient in water containing 0.5% trifluoroacetic acid and detected by their absorption at 405 nm. The positions of heme standards are indicated above the elution peaks.

 

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  		TABLE I
Purification steps of cytochrome bo3-like from B. cereus PYM1

 

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  		TABLE II
Some properties of the quinol oxidase bo3-like from B. cereus PYM1

 

Characterization of the bo3-like Oxidase from Bacillus cereus PYM1—SDS-PAGE analysis (Fig. 2A) of the purified cytochrome bo3-like showed two subunits with apparent molecular masses of 51 and 30 kDa, respectively. This result resembles that of the cytochrome oxidase aa3 subunit composition found in B. cereus wild type strain (i.e. 51 and 30 kDa (21)).



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  		FIG. 2.
Electrophoretic Analysis (SDS-PAGE) and Western immunoblotting of the purified bo3-like oxidase. 30 µg of protein was loaded in each lane. After electrophoresis the gel was split in two halves; one was for Coomassie Blue protein staining (A), and the other one was transferred to a nitrocellulose membrane for Western blotting analysis (B). The membrane was revealed with antibodies raised against the aa3 cytochrome oxidase of B. cereus.

 

Apoprotein Analysis—Because the apparent molecular masses of the subunits of the purified cytochrome bo3 of B. cereus PYMI were closer to those reported for the quinol oxidase aa3 of B. cereus (i.e. 51 and 30 kDa (21)) than to those reported for a typical cytochrome bo3 of E. coli (i.e. 58, 33, 22, and 17 kDa) (27)), we decided to test whether these polypeptides were indeed aa3 subunits by carrying out Western immunoblotting analyses using anti-aa3 antibodies. As shown in Fig. 2, the two bands present in the SDS-PAGE (Fig. 2A) reacted to the anti-aa3 antibodies (Fig. 2B). These results suggest that the apoprotein present in the purified cytochrome bo3-like from PYM1 strain is in fact the apoprotein from cytochrome oxidase aa3. Importantly, Coomassie staining and Western analyses were consistent with a 1:1 stoichiometry (Fig. 2).

Amino-terminal Sequence—To further confirm the identity of the purified bo3-like oxidase, the amino-terminal sequences of the 51- and 30-kDa subunits of both wild type aa3- and PYM1 bo3-oxidases were determined and compared. We failed to sequence the major subunits of both enzymes because their amino-terminal ends were blocked. By contrast, the sequence found for the amino-terminal ends of the 30-kDa subunits of both wild type and PYM enzymes were identical, LAVLNPQGPVAKXQYDLIV. The sequence is highly identical (i.e. 68–73%; www.ncbi.nlm/blast) to the homologous fragment comprising residues 26–46 of subunit II of quinol oxidases aa3 of B. subtilis, Bacillus halodurans, Listeria inocua, and Listeria monocytogenes; and more importantly, the sequence determined here is 94% identical to the homologous fragment present in the very recently released genome sequence of B. cereus AATCC14579 (GenBankTM accession number AE016877 [GenBank] ). This result demonstrates unequivocally that the cytochrome bo3 purified here is indeed a promiscuous variant of the aa3 apocytochrome of B. cereus.

Photodissociation Spectra Analyses—CO-complex spectra (as in Fig. 1B) reveal all CO-reactive hemoproteins, including hydroperoxidases and globins as well as putative oxidases. Terminal oxidases in general exhibit relatively slow CO recombination kinetics at subzero temperatures. Accordingly, the dissociated CO is trapped by a Cu proximal to the heme Fe to which CO has been attached (6) allowing the recording of the photodissociation spectra. Here the cytochrome bo3-CO complex was photodissociated at 77 K according to experimental procedures. The resulting spectrum showed troughs at 415, 553, and 563 nm; two peaks were present, a prominent peak at 430 nm and the other at 557 nm (Fig. 3). These absorbance maxima arise from the generation of the unligated, reduced cytochrome following dissociation of CO. The spectrum was stable with time, indicating a negligible slow recombination of CO at this cryogenic temperature. Such spectral features and a slow CO recombination kinetics have been reported for cytochrome bo3 in E. coli (24), and cytochrome bo3-like of B. cereus (17) where the half-times for CO recombination at 168 K were 47 min and 22 min, respectively.



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  		FIG. 3.
Photodissociation spectrum of the CO adduct of the purified bo3-like oxidase. The purified enzyme (50 µg of protein) was reduced with dithionite and bubbled with CO for 5 min before freezing in liquid nitrogen. The sample was photodissociated by exposing the preparation to three flashes of a photographic flash.

 

Catalytic Activity—Pure preparations of cytochrome bo3 were able to oxidize quinol (TCHQ) but failed to oxidize ferrocytochrome c (neither yeast nor horse), revealing it as a quinol oxidase. The enzyme showed a hyperbolic kinetic pattern of response to increasing concentrations of TCHQ with an apparent Km for reduced TCHQ of 498 µM and a Vmax of 21 µmol of O2 min–1mg–1, and the calculated turnover with TCHQ was 55 s–1 (Table II). The turnover value is within the reported range for other bacterial oxidases like the co3 oxidase of Methylophilus methylotrophus (21 s–1 (28)), the promiscuous cao3 oxidase of Bacillus PS3 (122 s–1 (4)), the novel b(o/a)3 oxidase of B. stearothermophilus (190 s–1 (8)), and remarkably, the aa3 oxidase of B. cereus (100 s–1 (21)). The quinol oxidase activity was inhibited by KCN with an I50 of 24 µM, nearly the reported sensitivity of the original oxidase aa3 of B. cereus wild type strain (10 µM (21)) and the novel oxidase b(o/a)3 of B. stearothermophilus (19 µM (8)). On the other hand, 2-n-heptyl 4-hydroxyquinoline-N-oxide fully inhibited the TCHQ oxidase activity of the purified bo3-like cytochrome. The calculated I50 for 2-n-heptyl 4-hydroxyquinoline-N-oxide was 300 µM, a concentration significantly lower than that required to inhibit (i.e. Ki = 1.7 mM) the ubiquinone-1 oxidase activity of cytochrome bo3 of E. coli (29).

Heme O in B. cereus Wild Type—Thus far, our results demonstrate that in the PYM1 spontaneous mutant lacking heme A, the aa3 apoprotein has the ability to carry hemes B and O, resulting in a functional promiscuous bo3-like cytochrome. Our previous studies in B. cereus wild type strain showed that cells obtained from well aerated cultures lack heme O (16). In addition, studies in Bacillus PS3 showed that only under O2-limited cultures, cytochrome o3 replaces cytochrome a3 in the O2-reducing site of the oxidase caa3 (2). Therefore it was interesting to know whether or not heme O is accumulating under O2-limiting culture conditions in B. cereus wild type strain, thus enabling the promiscuous assembly of cytochrome o-like oxidases.

HPLC analyses confirmed the presence of hemes B (retention time, 27.5 min) and A (retention time, 32 min) in membranes of wild type cells grown under vigorous aeration (Fig. 4A). No heme O (retention time, 34 min) was detected in these membranes. On the other hand, membranes obtained from cells grown under O2-limited conditions showed a different picture (Fig. 4B): Although heme B was present in a concentration comparable with that of cells from well aerated cultures, the concentration of heme A was significantly lower. Indeed, heme A is seen as a small shoulder in the new peak formed by heme O. A ratio of 1:0.13 was calculated for the concentrations of heme O and A, respectively, in cells obtained from O2-limited cultures. The above results support our prediction that the decrease in the oxygen availability promotes the accumulation of heme O and probably the promiscuity of cytochromes originally containing heme A.



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  		FIG. 4.
Reverse-phase HPLC analysis of membrane-associated hemes extracted from B. cereus wild type grown under O2-sufficient (A) and O2-limited (B) conditions. Hemes were extracted from membranes with acetone-HCl. Heme standards and hemes extracted from B. cereus membranes were eluted from the column by acetonitrile gradient in water containing 0.5% trifluoroacetic acid and detected by absorption at 405 nm.

 


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Many reports have suggested that the promiscuous assembly of terminal oxidases may be a general phenomenon in bacteria, promoted by different culture conditions or as a consequence of specific mutations (2–10). In most cases, hemes O and A replace each other in the high spin site of the binuclear center, producing functional enzymes with somewhat different kinetic properties (2, 4, 18). We have demonstrated that the membrane-bound bo3-like oxidase purified here has a very high affinity for oxygen (Km = 8.4 nM; (17)). Promiscuous replacements in the low spin site of terminal oxidases have been reported only in E. coli, where the heme B associated to the oxidase bo3 can be replaced by heme O to produce a functional oxidase oo3 (6).

To our knowledge, this is the first report of an active promiscuous oxidase resulting from the simultaneous substitution of its original hemes in the high and low spin sites. Therefore, it is highly relevant that the bo3-like oxidase purified from B. cereus PYM1 strain was in fact a promiscuous oxidase related to the original aa3 enzyme characterized previously (21). This conclusion is supported by their similar oligomeric structure, the inmunoreactivity of the bo3 subunits against the B. cereus aa3 antiserum, and more importantly, the identical NH2-terminal sequences including the first 19 residues of the 30-kDa subunits of both enzymes.

According to the spectroscopic analyses (Figs. 1 and 3, Table I), the purified bo3-like oxidase contains one molecule of each of cytochromes b and o. Because the suitability of heme O replacing heme A in the high spin site has been documented for several oxidases (2–5, 8), and the fact that we have previously provided spectral evidence to demonstrate the reaction of cytochrome o with oxygen in membranes of the PYM1 strain (17), we propose that the promiscuous enzyme purified here bears cytochrome o in the oxygen reduction site and therefore that cytochrome b should be bound to the low spin site. The occupation of the low spin site by heme B in the PYM1 apocytochrome aa3 is rather unexpected. There are no previous reports showing an enzymatically active promiscuous oxidase in which heme B replaces the original heme A in either the low or high spin sites. The hydroxy-farnesyl-ethyl group of heme A in the low spin site is almost in the extended conformation, acting as a hydrophobic anchor held by the helical structure of the enzyme (30, 31). This interaction is an important structural feature but one that seems not to be crucial for the activity of the PYM1 promiscuous cytochrome bo3.

The wild type strain of B. cereus contains two terminal oxidases bearing heme A: cytochromes aa3 and caa3 (21, 22). Once solubilized from membranes, these cytochromes are readily separated each from other by an anionic-exchange chromatography step, because the caa3 enzyme is not retained by the column (22). Although we demonstrated previously that the 37.5-kDa cytochrome c-subunit of the caa3 enzyme is present in the membranes of PYM 1 strain in amounts comparable to those detected in the wild type strain (16), we might conclude that a functional promiscuous variant of the caa3 apocytochrome seems not to be present in the PYM1 strain based on the following: 1) we could not confirm the presence of oxidase activity (TMPD oxidase) attributable to a promiscuous variant (i.e. cbo3) of the original caa3 enzyme; and 2) heme O was not detected by HPLC in the chromatographic fractions that contained cytochrome c (not shown). Perhaps the double heme substitution is not functionally permissive for the caa3 apocytochrome. The purification and prosthetic group analyses of this enzyme from the PYM1 strain warrant further study.

It has been shown that in B. subtilis as well as in B. cereus the oxidase caa3 is induced during early stages of sporulation (22, 32). It seems that this oxidase is mainly responsible for the respiration burst observed during early sporulation stages and the rapid removal of organic acids accumulated during growth in fermentable media, thus leading to avoidance of excessive medium acidification. The lack of an active promiscuous variant of the oxidase caa3 in B. cereus PYM1 strain would explain the phenotype reported earlier (16), consisting of low TMPD oxidase activity, lack of a respiration burst, an excessive acidification of the medium, and the consequent asporogenic behavior shown in normal fermentable media. Raising the buffer capacity of the media restores normal growth and recovery of sporulation to levels comparable with the wild type strain (16).

Interestingly, B. cereus wild type strain grown under microaerophilic conditions displayed a severe decrease in the concentration of membrane-bound heme A, compensated for by an accumulation of heme O (Fig. 4). This condition would promote the assembly of promiscuous variants of the original cytochromes aa3 and caa3, containing different heme formulae, depending on the availability of heme A. Consistently it has been shown that in the Bacillus PS3, O2-limited growth promotes the promiscuous assembly of the enzymatically active cytochrome cao3 instead of the original cytochrome caa3. Here, we have demonstrated that under the extreme condition of lack of heme A, the aa3 oxidase is replaced by its active and promiscuous bo3 variant. However, under physiological conditions in which O2 becomes limited, a dynamic variation in the promiscuous assembly of oxidases may be a common event resulting in some variants becoming inactive but others possessing kinetic properties more suitable for microaerophilic life.


   FOOTNOTES
 
* This work was supported in part by Grants DGAPA-UNAM IN-215801 and CONACYT 34300-N. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

|| To whom correspondence should be addressed: Dept. Bioquímica, Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, Apdo. Postal 70-242, Mexico City 04510, Mexico. Tel.: 52-55-56225627; Fax: 52-55-56225630; E-mail: eescami{at}ifisiol.unam.mx.

1 The abbreviations used are: TMPD, N,N,N',N'-tetramethyl-p-phenylendiamine; TCHQ, tetrachorohydroquinol; HPLC, high-pressure liquid chromatography. Back


   ACKNOWLEDGMENTS
 
We are grateful to Nancy Méndez Jacobo, Enrique Gutiérrez, and Juan Manuel Méndez Franco for technical assistance and to Virginia Godínez for secretarial assistance.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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