A Novel Double Heme Substitution Produces a Functional bo 3 Variant of the Quinol Oxidase aa 3 of Bacillus cereus
2003; Elsevier BV; Volume: 278; Issue: 34 Linguagem: Inglês
10.1074/jbc.m302583200
ISSN1083-351X
AutoresMartha Lucinda Contreras-Zentella, Guillermo Mendoza, Jorge Membrillo‐Hernández, J. E. Escamilla,
Tópico(s)Electrochemical sensors and biosensors
ResumoA novel bo 3-type quinol oxidase was highly purified from Bacillus cereus PYM1, a spontaneous mutant unable to synthesize heme A and therefore spectroscopically detectable cytochromes aa 3 and caa 3. 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 M r of 51,000 and 30,000, respectively. Both subunits were immunoreactive to antibodies raised against the B cereus aa 3 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 aa 3 oxidase. The purified bo 3 oxidase failed to oxidize ferrrocytochrome c (neither yeast nor horse) but oxidized tetrachlorohydroquinol with an apparent K m of 498 μm, a V max 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 bo 3 oxidase of this mutant is not the product of a new operon but instead is a cytochrome aa 3 apoprotein encoded by the qox operon of the aa 3 oxidase of B. cereus wild type promiscuously assembled with hemes B and O replacing heme A, producing a novel bo 3 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. A novel bo 3-type quinol oxidase was highly purified from Bacillus cereus PYM1, a spontaneous mutant unable to synthesize heme A and therefore spectroscopically detectable cytochromes aa 3 and caa 3. 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 M r of 51,000 and 30,000, respectively. Both subunits were immunoreactive to antibodies raised against the B cereus aa 3 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 aa 3 oxidase. The purified bo 3 oxidase failed to oxidize ferrrocytochrome c (neither yeast nor horse) but oxidized tetrachlorohydroquinol with an apparent K m of 498 μm, a V max 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 bo 3 oxidase of this mutant is not the product of a new operon but instead is a cytochrome aa 3 apoprotein encoded by the qox operon of the aa 3 oxidase of B. cereus wild type promiscuously assembled with hemes B and O replacing heme A, producing a novel bo 3 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. Bacteria have exploited unique terminal oxidases depending on the natural habitats and modes of aerobic metabolism (1Anraku Y. Annu. Rev. Biochem. 1988; 57: 101-132Crossref PubMed Scopus (171) Google Scholar). 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 (1Anraku Y. Annu. Rev. Biochem. 1988; 57: 101-132Crossref PubMed Scopus (171) Google Scholar). Additionally, terminal oxidases contain different heme prosthetic groups that have provided a customary way of identification (i.e. aa 3, bo 3, caa 3, bd, cbb 3, ba 3). However this classification is further complicated by the fact that under specific culture conditions (2Sone N. Kutoh E. Sato K. J. Biochem. 1990; 107: 547-602Crossref Scopus (12) Google Scholar, 3Quereshi M.H. Yumoto I. Fujiwara T. Fukumori Y. J. Biochem. 1990; 107: 408-485Google Scholar, 4Sone N. Fujiwara Y. FEBS Lett. 1991; 288: 154-158Crossref PubMed Scopus (54) Google Scholar, 5Matsushita K. Ebisuya H. Ameyama M. Adachi O. J. Bacteriol. 1992; 174: 122-129Crossref PubMed Google Scholar) or as result of mutations (6Puustinen A. Morgan J.E. Verkhovsky M. Thomas J.W. Gennis R.B. Wikström M. Biochemistry. 1992; 31: 10363-10369Crossref PubMed Scopus (79) Google Scholar, 7Zickermann I. Tautu O.S. Link T.A. Korn M. Ludwing B. Richter H. Eur. J. Biochem. 1997; 246: 618-624Crossref PubMed Scopus (16) Google Scholar, 8Sakamoto J. Handa Y. Sone N. J. Biochem. 1997; 122: 764-771Crossref PubMed Scopus (26) Google Scholar, 9Azarkina N. Siletsky S. Borisov V. von Wachenfeldt C. Hederstedt L. Konstantinov A.A. J. Biol. Chem. 1999; 274: 32810-32817Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 10Hill J. Groswitz V. Calhoun M. García-Horsman A. Lemieux L. Alben J.O. Gennis R.B. Biochemistry. 1992; 31: 11435-11440Crossref PubMed Scopus (61) Google Scholar), 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 bo 3 of over-expressing strains of Escherichia coli, resulting in the assembly of a functional cytochrome, oo 3. It is noteworthy that the heme of the binuclear O2 reduction site was invariably heme O (6Puustinen A. Morgan J.E. Verkhovsky M. Thomas J.W. Gennis R.B. Wikström M. Biochemistry. 1992; 31: 10363-10369Crossref PubMed Scopus (79) Google Scholar). 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 bb 3 enzyme (10Hill J. Groswitz V. Calhoun M. García-Horsman A. Lemieux L. Alben J.O. Gennis R.B. Biochemistry. 1992; 31: 11435-11440Crossref PubMed Scopus (61) Google Scholar). Similarly Zikermann et al. (7Zickermann I. Tautu O.S. Link T.A. Korn M. Ludwing B. Richter H. Eur. J. Biochem. 1997; 246: 618-624Crossref PubMed Scopus (16) Google Scholar) reported that in Paracoccus denitrificans ctaB-deleted strains (the ctaB gene is an orthologue of cyoE), a bb 3 variant of bo 3 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 caa 3-type oxidase (8Sakamoto J. Handa Y. Sone N. J. Biochem. 1997; 122: 764-771Crossref PubMed Scopus (26) Google Scholar). Likewise, the presence of a b(a/o)3-type oxidase was reported in Acetobacter aceti (5Matsushita K. Ebisuya H. Ameyama M. Adachi O. J. Bacteriol. 1992; 174: 122-129Crossref PubMed Google Scholar), a ca(a/o)3-type oxidase was found in Bacillus PS3 (4Sone N. Fujiwara Y. FEBS Lett. 1991; 288: 154-158Crossref PubMed Scopus (54) Google Scholar), and an a(a/o)3-type oxidase was isolated from cyanobacteria (11Peschek G.A. Alge D. Fromwald S. Meyer B. J. Biol. Chem. 1995; 270: 27937-27941Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar, 12Auer G. Mayer B. Wastyn M. Fromwald S. Eghbalzad K. Alge D. Peschek G.A. Biochem. Mol. Biol. Int. 1995; 37: 1173-1185PubMed Google Scholar). 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 (13Saiki K. Mogi T. Anraku Y. Biochem. Biophys. Res. Commun. 1992; 189: 1491-1497Crossref PubMed Scopus (84) Google Scholar, 14Svensson B. Andersson K.K. Hederstedt L. Eur. J. Biochem. 1996; 238: 287-295Crossref PubMed Scopus (43) Google Scholar). 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 (3Quereshi M.H. Yumoto I. Fujiwara T. Fukumori Y. J. Biochem. 1990; 107: 408-485Google Scholar, 4Sone N. Fujiwara Y. FEBS Lett. 1991; 288: 154-158Crossref PubMed Scopus (54) Google Scholar) 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 (14Svensson B. Andersson K.K. Hederstedt L. Eur. J. Biochem. 1996; 238: 287-295Crossref PubMed Scopus (43) Google Scholar)) as well as, in other genetically uncharacterized (presumably ctaA) mutants (B. subtilis FG83 (15James W.S. Gibson F. Taroni P. Poole R.K. FEMS Microbiol. Lett. 1989; 58: 277-282Crossref Scopus (16) Google Scholar) and Bacillus cereus PYM1 (16Del Arenal I.P. Contreras M.L. Svaeteorova B.B. Rangel O. Dávila J.R. Lledías F. Escamilla J.E. Arch. Microbiol. 1997; 167: 24-31Crossref PubMed Scopus (15) Google Scholar)). 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 (15James W.S. Gibson F. Taroni P. Poole R.K. FEMS Microbiol. Lett. 1989; 58: 277-282Crossref Scopus (16) Google Scholar, 17Contreras M.L. Escamilla J.E. Del Arenal P. Dávila J.R. D'Mello R. Poole R.K. Microbiology. 1999; 145: 1563-1573Crossref PubMed Scopus (11) Google Scholar). 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 (4Sone N. Fujiwara Y. FEBS Lett. 1991; 288: 154-158Crossref PubMed Scopus (54) Google Scholar, 6Puustinen A. Morgan J.E. Verkhovsky M. Thomas J.W. Gennis R.B. Wikström M. Biochemistry. 1992; 31: 10363-10369Crossref PubMed Scopus (79) Google Scholar, 18Matsushita K. Ebisuya H. Adachi O. J. Biol. Chem. 1992; 267: 24748-24753Abstract Full Text PDF PubMed Google Scholar). 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 bo 3 quinol oxidase from B. cereus PYM1. Our results indicate that the purified bo 3 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 aa 3 quinol oxidase apocytochrome. On the other hand, a functional promiscuous equivalent was not detected for the caa 3 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 bo 3 oxidase were analyzed and compared with those of the original aa 3 oxidase. Bacterial Strains and Growth Conditions—A B. cereus wild type strain originally isolated by Andreoli et al. (19Andreoli A.J. Suehiro S. Sakeyama D. Takemoto J. Vivanco E. Lara J.C. Klute M.C. J. Bacteriol. 1973; 115: 1159-1166Crossref PubMed Google Scholar) and its spontaneous derivative, PYM1 (16Del Arenal I.P. Contreras M.L. Svaeteorova B.B. Rangel O. Dávila J.R. Lledías F. Escamilla J.E. Arch. Microbiol. 1997; 167: 24-31Crossref PubMed Scopus (15) Google Scholar), 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 (20Hederstedt L. Methods Enzymol. 1986; 126: 399-414Crossref PubMed Scopus (61) Google Scholar) 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 (16Del Arenal I.P. Contreras M.L. Svaeteorova B.B. Rangel O. Dávila J.R. Lledías F. Escamilla J.E. Arch. Microbiol. 1997; 167: 24-31Crossref PubMed Scopus (15) Google Scholar). 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 bo 3-like oxidase from PYM1 strain was similar to those used for the purification of the B. cereus oxidases aa 3 and caa 3 reported earlier, with minor modifications (21García-Horsman J.A. Barquera B. González-Halphen D. Escamilla J.E. Mol. Microbiol. 1991; 5: 197-205Crossref PubMed Scopus (10) Google Scholar, 22García-Horsman J.A. Barquera B. Escamilla J.E. Eur. J. Biochem. 1991; 199: 761-768Crossref PubMed Scopus (14) Google Scholar). 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) 1The abbreviations used are: TMPD, N,N,N′,N′-tetramethyl-p-phenylendiamine; TCHQ, tetrachorohydroquinol; HPLC, high-pressure liquid chromatography. 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 (17Contreras M.L. Escamilla J.E. Del Arenal P. Dávila J.R. D'Mello R. Poole R.K. Microbiology. 1999; 145: 1563-1573Crossref PubMed Scopus (11) Google Scholar). Photodissociation Spectroscopy—The purified preparation of cytochrome bo 3-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 (23Poole R.K. Waring A.J. Chance B. Biochem. J. 1979; 184: 379-389Crossref PubMed Scopus (46) Google Scholar, 24Poole R.K. Salmon I. Chance B. Microbiology. 1994; 140: 1027-1034Crossref PubMed Scopus (17) Google Scholar). Electrophoretic and Western Blot Analyses—To decrease Triton X-100 content in purified preparations of cytochrome bo 3-like, samples were passed through a 0.3 × 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 (16Del Arenal I.P. Contreras M.L. Svaeteorova B.B. Rangel O. Dávila J.R. Lledías F. Escamilla J.E. Arch. Microbiol. 1997; 167: 24-31Crossref PubMed Scopus (15) Google Scholar). 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 aa 3 as described previously (16Del Arenal I.P. Contreras M.L. Svaeteorova B.B. Rangel O. Dávila J.R. Lledías F. Escamilla J.E. Arch. Microbiol. 1997; 167: 24-31Crossref PubMed Scopus (15) Google Scholar) Amino-terminal Sequence—Purified aa 3- and bo 3-like oxidases from wild type and PYM1 strains, respectively, were subjected to SDS-PAGE and electrophoretic transfer of proteins onto polyvinylidene difluoride membranes (25Towbin H. Staehelin T. Gordon J. Proc. Natl. Acad. Sci. U. S. A. 1979; 70: 4350-4354Crossref Scopus (44924) Google Scholar). Both proteins were sequenced by automated Edman degradation using a Beckman Spherogel Micro PTH (2 × 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 (16Del Arenal I.P. Contreras M.L. Svaeteorova B.B. Rangel O. Dávila J.R. Lledías F. Escamilla J.E. Arch. Microbiol. 1997; 167: 24-31Crossref PubMed Scopus (15) Google Scholar), 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 × 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 (16Del Arenal I.P. Contreras M.L. Svaeteorova B.B. Rangel O. Dávila J.R. Lledías F. Escamilla J.E. Arch. Microbiol. 1997; 167: 24-31Crossref PubMed Scopus (15) Google Scholar). 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. (26Markwell M.A.K. Hass S.M. Tolbert N.E. Bieber L.L. Methods Enzymol. 1981; 72: 296-303Crossref PubMed Scopus (724) Google Scholar) using bovine serum albumin as standard. Purification of the bo 3 -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 × 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 (22García-Horsman J.A. Barquera B. Escamilla J.E. Eur. J. Biochem. 1991; 199: 761-768Crossref PubMed Scopus (14) Google Scholar), further purification of this fraction confirmed the presence of cytochrome caa 3 (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 bo 3-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 bo 3 -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 bo 3-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 bo 3 -like oxidase of B. cereus PYM1. The results of the purification are summarized in Table I. The chromatographic behavior described for cytochrome bo 3-like of the PYM1 strain was similar to that described previously for a cytochrome aa 3 of the wild type strain (21García-Horsman J.A. Barquera B. González-Halphen D. Escamilla J.E. Mol. Microbiol. 1991; 5: 197-205Crossref PubMed Scopus (10) Google Scholar, 22García-Horsman J.A. Barquera B. Escamilla J.E. Eur. J. Biochem. 1991; 199: 761-768Crossref PubMed Scopus (14) Google Scholar).Table IPurification steps of cytochrome bo3-like from B. cereus PYM1StepProteinHeme OTCHQ oxidaseaTCHQ was reduced by ascorbate.Yield, oxidase activityPurification, heme OYield, heme Omgnmol-1 mg protein-1ng atoms O2 min-1 mg protein-1%-fold%Membranes42000.182501001100Triton X-100 extraction7706.6DEAE-Sepharose CL6B column1042.567571434QAE Toyopearl column294.6320082218HA Ultrogel column3.512.4158605696a TCHQ was reduced by ascorbate. Open table in a new tab Table IISome properties of the quinol oxidase bo 3-like from B. cereus PYM1Content of heme O12.4 nmol mg-1Content of heme B11.5 nmol mg-1Apparent Km for O28.4 nmApparent Km for TCHQ498 μmV max with TCHQ21 μmol of O2 min-1 mg-1Turnover with TCHQ55 s-1I50 for KCN24 μmI50 for HOQNOa2-n-heptyl 4-hydroxyquinoline-N-oxide.300 μma 2-n-heptyl 4-hydroxyquinoline-N-oxide. Open table in a new tab Characterization of the bo 3 -like Oxidase from Bacillus cereus PYM1—SDS-PAGE analysis (Fig. 2A) of the purified cytochrome bo 3-like showed two subunits with apparent molecular masses of 51 and 30 kDa, respectively. This result resembles that of the cytochrome oxidase aa 3 subunit composition found in B. cereus wild type strain (i.e. 51 and 30 kDa (21García-Horsman J.A. Barquera B. González-Halphen D. Escamilla J.E. Mol. Microbiol. 1991; 5: 197-205Crossref PubMed Scopus (10) Google Scholar)). Apoprotein Analysis—Because the apparent molecular masses of the subunits of the purified cytochrome bo 3 of B. cereus PYMI were closer to those reported for the quinol oxidase aa 3 of B. cereus (i.e. 51 and 30 kDa (21García-Horsman J.A. Barquera B. González-Halphen D. Escamilla J.E. Mol. Microbiol. 1991; 5: 197-205Crossref PubMed Scopus (10) Google Scholar)) than to those reported for a typical cytochrome bo 3 of E. coli (i.e. 58, 33, 22, and 17 kDa) (27Carter-Minghetti K. Chepuri-Goswitz V. Gabriel N.E. Hill J.J. Barassi C.A. Georgiou C.D. Chan S.I. Gennis R.B. Biochemistry. 1992; 31: 6917-6924Crossref PubMed Scopus (62) Google Scholar)), we decided to test whether these polypeptides were indeed aa 3 subunits by carrying out Western immunoblotting analyses using anti-aa 3 antibodies. As shown in Fig. 2, the two bands present in the SDS-PAGE (Fig. 2A) reacted to the anti-aa 3 antibodies (Fig. 2B). These results suggest that the apoprotein present in the purified cytochrome bo 3-like from PYM1 strain is in fact the apoprotein from cytochrome oxidase aa 3. 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 bo 3-like oxidase, the amino-terminal sequences of the 51- and 30-kDa subunits of both wild type aa 3- and PYM1 bo 3-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 aa 3 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 (GenBank™ accession number AE016877). This result demonstrates unequivocally that the cytochrome bo 3 purified here is indeed a promiscuous variant of the aa 3 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 (6Puustinen A. Morgan J.E. Verkhovsky M. Thomas J.W. Gennis R.B. Wikström M. Biochemistry. 1992; 31: 10363-10369Crossref PubMed Scopus (79) Google Scholar) allowing the recording of the photodissociation spectra. Here the cytochrome bo 3-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 bo 3 in E. coli (24Poole R.K. Salmon I. Chance B. Microbiology. 1994; 140: 1027-1034Crossref PubMed Scopus (17) Google Scholar), and cytochrome bo 3 -like of B. cereus (17Contreras M.L. Escamilla J.E. Del Arenal P. Dávila J.R. D'Mello R. Poole R.K. Microbiology. 1999; 145: 1563-1573Crossref PubMed Scopus (11) Google Scholar) where the half-times for CO recombination at 168 K were 47 min and 22 min, respectively. Catalytic Activity—Pure preparations of cytochrome bo 3 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 K m for reduced TCHQ of 498 μm and a V max 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 co 3 oxidase of Methylophilus methylotrophus (21 s–1 (28Froud S. Anthony C. J. Gen. Microbiol. 1984; 130: 2201-2212Google Scholar)), the promiscuous cao 3 oxidase of Bacillus PS3 (122 s–1 (4Sone N. Fujiwara Y. FEBS Lett. 1991; 288: 154-158Crossref PubMed Scopus (54) Google Scholar)), the novel b(o/a)3 oxidase of B. stearothermophilus (190 s–1 (8Sakamoto J. Handa Y. Sone N. J. Biochem. 1997; 122: 764-771Crossref PubMed Scopus (26) Google Scholar)), and remarkably, the aa 3 oxidase of B. cereus (100 s–1 (21García-Horsman J.A. Barquera B. González-Halphen D. Escamilla J.E. Mol. Microbiol. 1991; 5: 197-205Crossref PubMed Scopus (10) Google Scholar)). The quinol oxidase activity was inhibited by KCN with an I50 of 24 μm, nearly the reported sensitivity of the original oxidase aa 3 of B. cereus wild type strain (10 μm (21García-Horsman J.A. Barquera B. González-Halphen D. Escamilla J.E. Mol. Microbiol. 1991; 5: 197-205Crossref PubMed Scopus (10) Google Scholar)) and the novel oxidase b(o/a)3 of B. stearothermophilus (19 μm (8Sakamoto J. Handa Y. Sone N. J. Biochem. 1997; 122: 764-771Crossref PubMed Scopus (26) Google Scholar)). On the other hand, 2-n-heptyl 4-hydroxyquinoline-N-oxide fully inhibited the TCHQ oxidase activity of the purified bo 3-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. K i = 1.7 mm) the ubiquinone-1 oxidase activity of cytochrome bo 3 of E. coli (29Sato-Watabe M. Mogi T. Miyoshi H. Anraku Y. Biochemistry. 1998; 37: 5356-5361Crossref PubMed Scopus (38) Google Scholar). Heme O in B. cereus Wild Type—Thus far, our results demonstrate that in the PYM1 spontaneous mutant lacking heme A, the aa 3 apoprotein has the ability to carry hemes B and O, resulting in a functional promiscuous bo 3-like cytochrome. Our previous studies in B. cereus wild type strain showed that cells obtained from well aerated cultures lack heme O (16Del Arenal I.P. Contreras M.L. Svaeteorova B.B. Rangel O. Dávila J.R. Lledías F. Escamilla J.E. Arch. Microbiol. 1997; 167: 24-31Crossref PubMed Scopus (15) Google Scholar). In addition, studies in Bacillus PS3 showed that only under O2-limited cultures, cytochrome o 3 replaces cytochrome a 3 in the O2-reducing site of the oxidase caa 3 (2Sone N. Kutoh E. Sato K. J. Biochem. 1990; 107: 547-602Crossref Scopus (12) Google Scholar). 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. 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 (2Sone N. Kutoh E. Sato K. J. Biochem. 1990; 107: 547-602Crossref Scopus (12) Google Scholar, 3Quereshi M.H. Yumoto I. Fujiwara T. Fukumori Y. J. Biochem. 1990; 107: 408-485Google Scholar, 4Sone N. Fujiwara Y. FEBS Lett. 1991; 288: 154-158Crossref PubMed Scopus (54) Google Scholar, 5Matsushita K. Ebisuya H. Ameyama M. Adachi O. J. Bacteriol. 1992; 174: 122-129Crossref PubMed Google Scholar, 6Puustinen A. Morgan J.E. Verkhovsky M. Thomas J.W. Gennis R.B. Wikström M. Biochemistry. 1992; 31: 10363-10369Crossref PubMed Scopus (79) Google Scholar, 7Zickermann I. Tautu O.S. Link T.A. Korn M. Ludwing B. Richter H. Eur. J. Biochem. 1997; 246: 618-624Crossref PubMed Scopus (16) Google Scholar, 8Sakamoto J. Handa Y. Sone N. J. Biochem. 1997; 122: 764-771Crossref PubMed Scopus (26) Google Scholar, 9Azarkina N. Siletsky S. Borisov V. von Wachenfeldt C. Hederstedt L. Konstantinov A.A. J. Biol. Chem. 1999; 274: 32810-32817Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 10Hill J. Groswitz V. Calhoun M. García-Horsman A. Lemieux L. Alben J.O. Gennis R.B. Biochemistry. 1992; 31: 11435-11440Crossref PubMed Scopus (61) Google Scholar). 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 (2Sone N. Kutoh E. Sato K. J. Biochem. 1990; 107: 547-602Crossref Scopus (12) Google Scholar, 4Sone N. Fujiwara Y. FEBS Lett. 1991; 288: 154-158Crossref PubMed Scopus (54) Google Scholar, 18Matsushita K. Ebisuya H. Adachi O. J. Biol. Chem. 1992; 267: 24748-24753Abstract Full Text PDF PubMed Google Scholar). We have demonstrated that the membrane-bound bo 3-like oxidase purified here has a very high affinity for oxygen (K m = 8.4 nm; (17Contreras M.L. Escamilla J.E. Del Arenal P. Dávila J.R. D'Mello R. Poole R.K. Microbiology. 1999; 145: 1563-1573Crossref PubMed Scopus (11) Google Scholar)). 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 bo 3 can be replaced by heme O to produce a functional oxidase oo 3 (6Puustinen A. Morgan J.E. Verkhovsky M. Thomas J.W. Gennis R.B. Wikström M. Biochemistry. 1992; 31: 10363-10369Crossref PubMed Scopus (79) Google Scholar). 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 bo 3-like oxidase purified from B. cereus PYM1 strain was in fact a promiscuous oxidase related to the original aa 3 enzyme characterized previously (21García-Horsman J.A. Barquera B. González-Halphen D. Escamilla J.E. Mol. Microbiol. 1991; 5: 197-205Crossref PubMed Scopus (10) Google Scholar). This conclusion is supported by their similar oligomeric structure, the inmunoreactivity of the bo 3 subunits against the B. cereus aa 3 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 bo 3-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 (2Sone N. Kutoh E. Sato K. J. Biochem. 1990; 107: 547-602Crossref Scopus (12) Google Scholar, 3Quereshi M.H. Yumoto I. Fujiwara T. Fukumori Y. J. Biochem. 1990; 107: 408-485Google Scholar, 4Sone N. Fujiwara Y. FEBS Lett. 1991; 288: 154-158Crossref PubMed Scopus (54) Google Scholar, 5Matsushita K. Ebisuya H. Ameyama M. Adachi O. J. Bacteriol. 1992; 174: 122-129Crossref PubMed Google Scholar, 8Sakamoto J. Handa Y. Sone N. J. Biochem. 1997; 122: 764-771Crossref PubMed Scopus (26) Google Scholar), 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 (17Contreras M.L. Escamilla J.E. Del Arenal P. Dávila J.R. D'Mello R. Poole R.K. Microbiology. 1999; 145: 1563-1573Crossref PubMed Scopus (11) Google Scholar), 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 aa 3 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 (30Tsukihara T. Aoyama H. Yamashiota E. Tomizaki T. Yamaguchi H. Shinzawa-Itoh K. Nakashima R. Yaono R. Yoshikawa S. Science. 1995; 269: 1069-1074Crossref PubMed Scopus (1292) Google Scholar, 31Riistama S. Verkhovsky M.I. Laakkonen L. Wikström M. Puustinen A. Biochim. Biophys. Acta. 2000; 1456: 1-4Crossref PubMed Scopus (19) Google Scholar). This interaction is an important structural feature but one that seems not to be crucial for the activity of the PYM1 promiscuous cytochrome bo 3. The wild type strain of B. cereus contains two terminal oxidases bearing heme A: cytochromes aa 3 and caa 3 (21García-Horsman J.A. Barquera B. González-Halphen D. Escamilla J.E. Mol. Microbiol. 1991; 5: 197-205Crossref PubMed Scopus (10) Google Scholar, 22García-Horsman J.A. Barquera B. Escamilla J.E. Eur. J. Biochem. 1991; 199: 761-768Crossref PubMed Scopus (14) Google Scholar). Once solubilized from membranes, these cytochromes are readily separated each from other by an anionic-exchange chromatography step, because the caa 3 enzyme is not retained by the column (22García-Horsman J.A. Barquera B. Escamilla J.E. Eur. J. Biochem. 1991; 199: 761-768Crossref PubMed Scopus (14) Google Scholar). Although we demonstrated previously that the 37.5-kDa cytochrome c-subunit of the caa 3 enzyme is present in the membranes of PYM 1 strain in amounts comparable to those detected in the wild type strain (16Del Arenal I.P. Contreras M.L. Svaeteorova B.B. Rangel O. Dávila J.R. Lledías F. Escamilla J.E. Arch. Microbiol. 1997; 167: 24-31Crossref PubMed Scopus (15) Google Scholar), we might conclude that a functional promiscuous variant of the caa 3 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. cbo 3) of the original caa 3 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 caa 3 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 caa 3 is induced during early stages of sporulation (22García-Horsman J.A. Barquera B. Escamilla J.E. Eur. J. Biochem. 1991; 199: 761-768Crossref PubMed Scopus (14) Google Scholar, 32Lauraeus M. Halüa T. Saraste M. Wikström M. Eur. J. Biochem. 1991; 197: 699-705Crossref PubMed Scopus (101) Google Scholar). 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 caa 3 in B. cereus PYM1 strain would explain the phenotype reported earlier (16Del Arenal I.P. Contreras M.L. Svaeteorova B.B. Rangel O. Dávila J.R. Lledías F. Escamilla J.E. Arch. Microbiol. 1997; 167: 24-31Crossref PubMed Scopus (15) Google Scholar), 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 (16Del Arenal I.P. Contreras M.L. Svaeteorova B.B. Rangel O. Dávila J.R. Lledías F. Escamilla J.E. Arch. Microbiol. 1997; 167: 24-31Crossref PubMed Scopus (15) Google Scholar). 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 aa 3 and caa 3, 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 cao 3 instead of the original cytochrome caa 3. Here, we have demonstrated that under the extreme condition of lack of heme A, the aa 3 oxidase is replaced by its active and promiscuous bo 3 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. We are grateful to Nancy Méndez Jacobo, Enrique Gutiérrez, and Juan Manuel Méndez Franco for technical assistance and to Virginia Godínez for secretarial assistance.
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