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• W1965481505 abstract "Respiration of Escherichia colicatalyzed either by cytochrome bo′ or bd is sensitive to micromolar extracellular NO; extensive, transient inhibition of respiration increases as dissolved oxygen tension in the medium decreases. At low oxygen concentrations (25–33 μm), the duration of inhibition of respiration by 9 μm NO is increased by mutation of either oxidase. Respiration of an hmp mutant defective in flavohemoglobin (Hmp) synthesis is extremely NO-sensitive (I50 about 0.8 μm); conversely, cells pre-grown with sodium nitroprusside or overexpressing plasmid-bornehmp + are insensitive to 60 μm NO and have elevated levels of immunologically detectable Hmp. Purified Hmp consumes O2 at a rate that is instantaneously and extensively (>10-fold) stimulated by NO due to NO oxygenase activity but, in the absence of NO, Hmp does not contribute measurably to cell oxygen consumption. Cyanide binds to Hmp (K d 3 μm). Concentrations of KCN (100 μm) that do not significantly inhibit cell respiration markedly suppress the protection of respiration from NO afforded by Hmp and abolish NO oxygenase activity of purified Hmp. The results demonstrate the role of Hmp in protecting respiration from NO stress and are discussed in relation to the energy metabolism of E. coli in natural O2-depleted environments. Respiration of Escherichia colicatalyzed either by cytochrome bo′ or bd is sensitive to micromolar extracellular NO; extensive, transient inhibition of respiration increases as dissolved oxygen tension in the medium decreases. At low oxygen concentrations (25–33 μm), the duration of inhibition of respiration by 9 μm NO is increased by mutation of either oxidase. Respiration of an hmp mutant defective in flavohemoglobin (Hmp) synthesis is extremely NO-sensitive (I50 about 0.8 μm); conversely, cells pre-grown with sodium nitroprusside or overexpressing plasmid-bornehmp + are insensitive to 60 μm NO and have elevated levels of immunologically detectable Hmp. Purified Hmp consumes O2 at a rate that is instantaneously and extensively (>10-fold) stimulated by NO due to NO oxygenase activity but, in the absence of NO, Hmp does not contribute measurably to cell oxygen consumption. Cyanide binds to Hmp (K d 3 μm). Concentrations of KCN (100 μm) that do not significantly inhibit cell respiration markedly suppress the protection of respiration from NO afforded by Hmp and abolish NO oxygenase activity of purified Hmp. The results demonstrate the role of Hmp in protecting respiration from NO stress and are discussed in relation to the energy metabolism of E. coli in natural O2-depleted environments. nitric oxide S-nitrosoglutathione S-nitrosocysteine sodium nitroprusside 4-morpholinepropanesulfonic acid The pervasive importance in biology of nitric oxide,i.e. the nitrogen monoxide radical, NO⋅, written here as NO,1 (1Bonner F.T. Hughes M.N. Comments Inorg. Chem. 1988; 7: 215-234Crossref Scopus (92) Google Scholar, 2Bonner F.T. Stedman G. Feelisch M. Stamler J.S. Methods in Nitric Oxide Research. Wiley, Chichester1996: 3-18Google Scholar) is now widely appreciated. NO plays key roles in vasodilation and intracellular signaling, yet is toxic (3Murad F. Biosci. Rep. 1999; 19: 133-154Crossref PubMed Scopus (88) Google Scholar). Microbiologically, NO is important as an intermediate in denitrification (4Watmough N.J. Butland G. Cheesman M.R. Moir J.W.B. Richardson D.J. Spiro S. Biochim. Biophys. Acta. 1999; 1411: 456-474Crossref PubMed Scopus (118) Google Scholar) and as a component of the antibacterial arsenal of reactive oxygen and nitrogen species generated in phagolysosomes (5De Groote M.A. Fang F.C. Clin. Infect. Dis. 1995; 21: S162-S165Crossref PubMed Scopus (277) Google Scholar). NO and redox-related species such as the nitrosonium cation (NO+) and the nitroxyl ion (NO−) (6Hughes M.N. Biochim. Biophys. Acta. 1999; 1411: 263-272Crossref PubMed Scopus (244) Google Scholar) are reactive with many key biomolecules including iron proteins (7Cooper C.E. Biochim. Biophys. Acta. 1999; 1411: 290-309Crossref PubMed Scopus (460) Google Scholar), copper proteins (8Torres J. Wilson M.T. Biochim. Biophys. Acta. 1999; 1411: 310-322Crossref PubMed Scopus (49) Google Scholar), and thiol groups (9Gaston B. Biochim. Biophys. Acta. 1999; 1411: 323-333Crossref PubMed Scopus (234) Google Scholar). NO is a well established and experimentally useful ligand for heme proteins; its reactions with globins and oxidases from diverse higher organisms have been extensively investigated but assumed to have little physiological significance (10Yoshikawa T. Ohya-Nishiguchi H. Packer L. Bioradicals Detected by ESR Spectroscopy. Birkhauser Verlag, Basel1995: 217-235Crossref Google Scholar). Recently, however, the hemoglobin-NO reaction has been shown to regulate the chemistry of NO and maintain it in a circulating bioactive state (11Gow A.J. Luchsinger B.P. Pawloski J.R. Singel D.J. Stamler J.S. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 9027-9032Crossref PubMed Scopus (378) Google Scholar). Reaction of NO with the mitochondrial terminal oxidase, cytochromeaa 3, has been shown to involve not only the O2-binding heme (cytochrome a 3) but also the copper atom (CuB) (8Torres J. Wilson M.T. Biochim. Biophys. Acta. 1999; 1411: 310-322Crossref PubMed Scopus (49) Google Scholar, 12Torres J. Cooper C.E. Sharpe M. Wilson M.T. J. Bioenerg. Biomem. 1998; 30: 63-69Crossref PubMed Scopus (17) Google Scholar) which together constitute the O2-reducing binuclear active site of the enzyme. E. coli does not have cytochromeaa 3 but synthesizes two other major respiratory quinol oxidases, cytochromes bo′ and bd (13Gennis R.B. Stewart V. Niedhardt F.C. Curtiss III, R. Ingraham J.L. Lin E.C.C. Low K.B. Magasanik B. Reznikoff W.S. Riley M. Schaechter M. Umbarger H.E. Escherichia coli and Salmonella Cellular and Molecular Biology. 2nd Ed. American Society for Microbiology Press, Washington, D. C.1996: 217-261Google Scholar, 14Poole R.K. Cook G.M. Adv. Microbial Physiol. 2000; 43: 165-224Crossref PubMed Google Scholar). Cytochrome bo′ is structurally and functionally homologous to mitochondrial cytochrome aa 3 and binds two molecules of NO at CuB (15Butler C.S. Seward H.E. Greenwood C. Thomson A.J. Biochemistry. 1997; 36: 16259-16266Crossref PubMed Scopus (73) Google Scholar). Cytochrome bdreacts with nitrite and trioxodinitrate to form a spectrally distinctive nitrosyl complex (16Hubbard J.A.M. Hughes M.N. Poole R.K. FEBS Lett. 1983; 164: 241-243Crossref PubMed Scopus (14) Google Scholar, 17Hubbard J.A.M. Hughes M.N. Poole R.K. Poole R.K. Dow C.S. Microbial Gas Metabolism-Mechanistic, Metabolic and Biotechnological Aspects. Academic Press, London1985: 231-236Google Scholar, 18Hori H. Tsubaki M. Mogi T. Anraku Y. J. Biol. Chem. 1996; 271: 9254-9258Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). Since cytochromes bo′and bd have distinct structures and catalytic properties, including sensitivity to cyanide (14Poole R.K. Cook G.M. Adv. Microbial Physiol. 2000; 43: 165-224Crossref PubMed Google Scholar), and are differentially regulated (13Gennis R.B. Stewart V. Niedhardt F.C. Curtiss III, R. Ingraham J.L. Lin E.C.C. Low K.B. Magasanik B. Reznikoff W.S. Riley M. Schaechter M. Umbarger H.E. Escherichia coli and Salmonella Cellular and Molecular Biology. 2nd Ed. American Society for Microbiology Press, Washington, D. C.1996: 217-261Google Scholar, 14Poole R.K. Cook G.M. Adv. Microbial Physiol. 2000; 43: 165-224Crossref PubMed Google Scholar), they might be expected to be differentially sensitive to NO, particularly since cytochrome bd lacks the NO-reactive copper site (13Gennis R.B. Stewart V. Niedhardt F.C. Curtiss III, R. Ingraham J.L. Lin E.C.C. Low K.B. Magasanik B. Reznikoff W.S. Riley M. Schaechter M. Umbarger H.E. Escherichia coli and Salmonella Cellular and Molecular Biology. 2nd Ed. American Society for Microbiology Press, Washington, D. C.1996: 217-261Google Scholar, 14Poole R.K. Cook G.M. Adv. Microbial Physiol. 2000; 43: 165-224Crossref PubMed Google Scholar). Indeed, such a discrimination has been reported in plant mitochondria (19Millar A.H. Day D.A. FEBS Lett. 1996; 398: 155-158Crossref PubMed Scopus (191) Google Scholar), in which NO inhibits oxygen consumption by cytochrome aa3 but not by the cyanide-insensitive, non-phosphorylating, alternative oxidase. To date only the respiration of Escherichia coli possessing both oxidases has been shown to be inhibited by NO (20Yu H. Sato E.F. Nagata K. Nishikawa M. Kashiba M. Arakawa T. Kobayashi K. Tamura T. Inoue M. FEBS Lett. 1997; 409: 161-165Crossref PubMed Scopus (56) Google Scholar). E. coli responds at the level of gene expression to NO, NO-releasing agents, and nitrosating compounds in at least two ways. Nunoshiba et al. (21Nunoshiba T. DeRojas T. Wishnok J.S. Tannenbaum S.R. Demple B. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 9993-9997Crossref PubMed Scopus (280) Google Scholar) have demonstrated that NO activates the soxRS two-component global regulatory system which results in increased synthesis of superoxide dismutase and other enzymes that provide protection from oxidative stress. However, the physiological function(s) of this system in resisting NO is unclear. Second, NO, nitrite, the nitrosating compound sodium nitroprusside (SNP) and S-nitrosoglutathione (GSNO) are potent inducers ofhmp gene expression in E. coli (22Poole R.K. Anjum M.F. Membrillo-Hernández J. Kim S.O. Hughes M.N. Stewart V. J. Bacteriol. 1996; 178: 5487-5492Crossref PubMed Scopus (208) Google Scholar, 23Membrillo-Hernández J. Coopamah M.D. Channa A. Hughes M.N. Poole R.K. Mol. Microbiol. 1998; 29: 1101-1112Crossref PubMed Scopus (82) Google Scholar) andMycobacterium tuberculosis (24Hu Y.M. Butcher P.D. Mangan J.A. Rajandream M.A. Coates A.R.M. J. Bacteriol. 1999; 181: 3486-3493Crossref PubMed Google Scholar). The E. coli gene product HMP is a flavohemoglobin first proposed by Poole et al. (22Poole R.K. Anjum M.F. Membrillo-Hernández J. Kim S.O. Hughes M.N. Stewart V. J. Bacteriol. 1996; 178: 5487-5492Crossref PubMed Scopus (208) Google Scholar) to detoxify NO. Recent studies support this proposal: mutants carrying a poorly defined deletion in the glnB-hmpregion of the genome are more sensitive to growth inhibition by NO than are control strains (25Gardner P.R. Gardner A.M. Martin L.A. Salzman A.L. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 10378-10383Crossref PubMed Scopus (497) Google Scholar). Such mutants are also unable to catalyze NAD(P)H-dependent NO consumption (25Gardner P.R. Gardner A.M. Martin L.A. Salzman A.L. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 10378-10383Crossref PubMed Scopus (497) Google Scholar, 26Hausladen A. Gow A.J. Stamler J.S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14100-14105Crossref PubMed Scopus (255) Google Scholar) and are compromised in inducible resistance to nitrosative stress exerted byS-nitrosocysteine (SNO-Cys) (26Hausladen A. Gow A.J. Stamler J.S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14100-14105Crossref PubMed Scopus (255) Google Scholar). Unequivocal evidence that the hmp gene itself is responsible for aerobic resistance to NO and nitrosative stress has come from construction of genetically defined hmp mutants. An E. coli K12hmp mutant (RKP4545) was shown to be hypersensitive to killing by GSNO and SNP (27Membrillo-Hernández J. Coopamah M.D. Anjum M.F. Stevanin T.M. Kelly A. Hughes M.N. Poole R.K. J. Biol. Chem. 1999; 274: 748-754Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar) and a Salmonella hmp mutant showed increased sensitivity to acidified nitrite andS-nitrosothiols (28Crawford M.J. Goldberg D.E. J. Biol. Chem. 1998; 273: 12543-12547Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar). The roles of Hmp in protecting growth and aconitase activity from NO (27Membrillo-Hernández J. Coopamah M.D. Anjum M.F. Stevanin T.M. Kelly A. Hughes M.N. Poole R.K. J. Biol. Chem. 1999; 274: 748-754Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar, 29Gardner P.R. Costantino G. Salzman A.L. J. Biol. Chem. 1998; 273: 26528-26533Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar) have been largely attributed to the NO oxygenase activity of the protein. NO is proposed to attack oxygenated Hmp (Fe(III)-O2) to form the relatively innocuous nitrate ion (25Gardner P.R. Gardner A.M. Martin L.A. Salzman A.L. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 10378-10383Crossref PubMed Scopus (497) Google Scholar, 26Hausladen A. Gow A.J. Stamler J.S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14100-14105Crossref PubMed Scopus (255) Google Scholar). The additional ability of Hmp to sequester NO and reduce it to N2O (30Kim S.O. Orii Y. Lloyd D. Hughes M.N. Poole R.K. FEBS Lett. 1999; 445: 389-394Crossref PubMed Scopus (142) Google Scholar) may explain the protective effects of Hmp observed during anaerobic or microaerobic growth (25Gardner P.R. Gardner A.M. Martin L.A. Salzman A.L. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 10378-10383Crossref PubMed Scopus (497) Google Scholar) and may be of greater importance in natural environments of E. coli, including the gut (31Poole R.K. Ingledew W.J. Neidhardt F.C. Ingraham J.L. Low K.B. Magasanik B. Schaechter M. Umbarger H.E. Escherichia coli and Salmonella typhimurium-Cellular and Molecular Biology. American Society for Microbiology Press, Washington, D. C.1987: 170-200Google Scholar). Thus, Hmp, a member of the ancient globin family, plays key roles in resisting NO in several bacteria and probably other microorganisms (32Poole R.K. Hughes M.N. Mol. Microbiol. 2000; 36: 775-783Crossref PubMed Scopus (304) Google Scholar). The aim of this work was to determine the sensitivity to NO of respiration catalyzed by each of the major respiratory oxidases ofE. coli, cytochromes bo′ and bd. We show that NO inhibits both oxidases but that expression and activity of Hmp provides effective protection in vivo. Since the natural environment of E. coli in the gut is microaerobic (31Poole R.K. Ingledew W.J. Neidhardt F.C. Ingraham J.L. Low K.B. Magasanik B. Schaechter M. Umbarger H.E. Escherichia coli and Salmonella typhimurium-Cellular and Molecular Biology. American Society for Microbiology Press, Washington, D. C.1987: 170-200Google Scholar), where NO inhibition of respiration is maximal (Ref. 20Yu H. Sato E.F. Nagata K. Nishikawa M. Kashiba M. Arakawa T. Kobayashi K. Tamura T. Inoue M. FEBS Lett. 1997; 409: 161-165Crossref PubMed Scopus (56) Google Scholar, this work), it is likely that Hmp plays a critical role in enterobacterial growth and survival. Strains and the plasmid used are listed in Table I. Transformations were done after CaCl2 treatment (35Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). Cells were grown in rich medium (TY) (36Maloy S.R. Stewart V.J. Taylor R.K. Genetic Analysis of Pathogenic Bacteria. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1996Google Scholar) supplemented as appropriate with kanamycin (50 μg/ml) or ampicillin (100 μg/ml). Culture optical density at 600 nm (A 600) was measured with a Jenway 6100 spectrophotometer in cells of 10-mm path-length after appropriate dilution. Cultures were grown at 37 °C with shaking (200 rpm) in baffled conical flasks containing about 1/5 of their own volume of medium, and inoculated with 1% of their volume using an overnight culture.Table IBacterial strains and plasmid used in this studyRelevant genotypeSource/Ref.Strains AN2342Wild-typeRef.33Poole R.K. Williams H.D. Downie J.A. Gibson F. J. Gen. Microbiol. 1989; 135: 1865-1874PubMed Google Scholar AN2343cydD1Ref. 33Poole R.K. Williams H.D. Downie J.A. Gibson F. J. Gen. Microbiol. 1989; 135: 1865-1874PubMed Google Scholar HW350cyo::KmRDr. Huw D. Williams RSC521Strain RSC49 transformed with pPL341Dr. Nick Dixon (34) RKP4544As AN2342 but transduced with Pl(cyo::KmR) from HW350Malini Coopamah RKP4545hmp::KmRRef. 27Membrillo-Hernández J. Coopamah M.D. Anjum M.F. Stevanin T.M. Kelly A. Hughes M.N. Poole R.K. J. Biol. Chem. 1999; 274: 748-754Abstract Full Text Full Text PDF PubMed Scopus (135) Google ScholarPlasmid pPL341hmp + cloned in pBR322 (ApR)Ref. 34Vasudevan S.G. Armarego W.L.F. Shaw D.C. Lilley P.E. Dixon N.E. Poole R.K. Mol. Gen. Genet. 1991; 226: 49-58Crossref PubMed Scopus (184) Google Scholar Open table in a new tab NO was prepared as in Ref. 22Poole R.K. Anjum M.F. Membrillo-Hernández J. Kim S.O. Hughes M.N. Stewart V. J. Bacteriol. 1996; 178: 5487-5492Crossref PubMed Scopus (208) Google Scholar and GSNO synthesized as described in Ref. 23Membrillo-Hernández J. Coopamah M.D. Channa A. Hughes M.N. Poole R.K. Mol. Microbiol. 1998; 29: 1101-1112Crossref PubMed Scopus (82) Google Scholar. SNP was from Sigma. Non-sterilized solutions of these species were added to a culture 1.5 h after inoculation; after 3 h further growth, the cultures were harvested and used to prepare cell extracts. E. coli strain RSC521 (with multicopy plasmid pPL341, having the wild-typehmp + gene under the control of its native promoter) (34Vasudevan S.G. Armarego W.L.F. Shaw D.C. Lilley P.E. Dixon N.E. Poole R.K. Mol. Gen. Genet. 1991; 226: 49-58Crossref PubMed Scopus (184) Google Scholar) was grown aerobically, disrupted in a French pressure cell, and used to purify Hmp by two chromatographic steps (37Ioannidis N. Cooper C.E. Poole R.K. Biochem. J. 1992; 288: 649-655Crossref PubMed Scopus (67) Google Scholar). An improved purification protocol was used for the Hmp employed in the experiment shown in Fig. 7. 2C. E. Mills, S. Sedelnikova, B. Søballe, M. N. Hughes, and R. K. Poole, submitted for publication.Enzyme concentration was measured from spectra of the native (ferric) enzyme using the absorption coefficients described by Ioannidiset al. (37Ioannidis N. Cooper C.E. Poole R.K. Biochem. J. 1992; 288: 649-655Crossref PubMed Scopus (67) Google Scholar). Rabbits were administered subcutaneous injections with a homogeneous mixture (250 μl) of 2.7 mg of purified Hmp in Freund's complete adjuvant. Subsequently, two booster injections of similar composition were administered at 3 and 7 weeks, respectively, after the first injection. At the end of the procedure, rabbits were sacrificed, and bled out for serum. E. coli cells were disrupted by ultrasonication using an MSE/Sanyo Soniprep 150 sonicator delivering three 30-s periods at full power, with 30-s cooling intervals. Extracts were clarified by centrifugation at 132,000 × g for 1.5 h at 4 °C and used in Western blots (39Renart J. Sandoval I.V. Methods Enzymol. 1984; 104: 455-460Crossref PubMed Scopus (51) Google Scholar); detection was done using the ECL chemiluminescence system (Amersham Pharmacia Biotech). Cells were grown for 6 h as above untilA 600 reached approximately 1.4. Cells were harvested by centrifugation, washed in 0.9% sterile saline, and resuspended in 5 ml of buffer containing HEPES (50 mm, pH 7.4), 100 mm NaCl, 5 mm KCl, and 1 mm each of MgCl2, NaH2PO4, d-glucose, and CaCl2 (20Yu H. Sato E.F. Nagata K. Nishikawa M. Kashiba M. Arakawa T. Kobayashi K. Tamura T. Inoue M. FEBS Lett. 1997; 409: 161-165Crossref PubMed Scopus (56) Google Scholar). A Clark-type polarographic oxygen electrode system (Rank Bros., Bottisham, Cambridge, UK) was used comprising a water-jacketed (37 °C) Perspex chamber stirred magnetically; the membrane-covered electrode was situated at the bottom of the chamber below the stirrer. About 25–50 μl of cell suspension was diluted in the chamber with buffer to give a working volume of 2 ml and a close-fitting lid, with a fine hole for injections using a Hamilton syringe, was inserted. The suspension was further supplemented with glucose (10 μm final concentration) and respiration rates measured in the closed system. Additions of anoxic, NO-saturated solutions were made in the same way. The electrode was calibrated with air-equilibrated water (taken to contain 220 μmO2) and on adding sodium dithionite to achieve anoxia. The above oxygen electrode system was modified to permit simultaneous measurements of O2 and NO consumption.2 Briefly, the chamber top was sealed with a Perspex cap having a concave bottom surface and drilled with a central vertical hole (6 mm in diameter to a depth of approximately 33 mm, and 2.5 mm diameter for the remaining 11-mm depth) to accept an ISO-NOP2 stainless steel shielded NO electrode of 2-mm diameter (World Precision Instruments). The electrode shaft was fitted with a plastic sleeve to hold the membrane at the required depth in the chamber. A second vertical hole (approximately 1.5-mm diameter, 44-mm depth) near the cap perimeter permitted addition of solutions of NADH, enzymes, and NO. Current from the O2 electrode was amplified and displayed on a two-channel recorder, along with the amplified current of the NO electrode (polarizing voltage of 0.865 V) processed using a World Precision Instruments ISO-NO Mark II Isolated Nitric Oxide Meter. Experiments with purified Hmp were performed in a buffer containing 50 mm MOPS and 50 mm NaCl, pH 7.0. Spectra for determining the cytochrome composition of mutant strains were obtained using an SDB-4 dual-wavelength scanning spectrophotometer (University of Pennsylvania Biomedical Instrumentation Group, and Current Designs Inc., Philadelphia, PA) (40Kalnenieks U. Galinina N. Bringer-Meyer S. Poole R.K. FEMS Microbiol. Lett. 1998; 168: 91-97PubMed Google Scholar). Cells were centrifuged from stationary phase cultures, suspended in 0.1 m potassium phosphate buffer, pH 7.4, and used to record dithionite-reduced minuspersulfate-oxidized difference spectra or CO + dithioniteminus dithionite difference spectra (41Poole R.K. Kalnenieks U. Gore M. Spectrophotometry and Spectrofluorimetry: A Practical Approach. Oxford University Press, Oxford2000: 1-32Google Scholar). Spectral data were analyzed and plotted using SoftSDB (Current Designs Inc.) and CA-Cricket Graph III software. To assess the presence or absence of cytochrome o′, photodissociation spectra (photolysed,i.e. reduced minus unphotolysed, i.e.CO-ligated) were obtained at −100 °C in the above apparatus (42Søballe B. Poole R.K. Microbiology. 1998; 144: 361-373Crossref PubMed Scopus (24) Google Scholar). Purified Hmp is largely in the ferric state (37Ioannidis N. Cooper C.E. Poole R.K. Biochem. J. 1992; 288: 649-655Crossref PubMed Scopus (67) Google Scholar). Protein (final concentration typically 3–4 μm) was incubated in 1 ml of 50 mm MOPS/NaOH buffer, pH 7.0, containing 50 μm potassium ferricyanide to ensure complete conversion to the ferric form. Cyanide was added as a solution of NaCN, adjusted to pH 7.0, to give the final concentrations shown under “Results” and the samples (1 ml final volume) incubated at 4 °C overnight. Transition of the high-spin form to the low-spin cyanide complex was measured as ΔA at 423–387 nm. Among the many possible targets for the cellular toxic effects of NO and related reactive species, respiratory heme enzymes are prime candidates (8Torres J. Wilson M.T. Biochim. Biophys. Acta. 1999; 1411: 310-322Crossref PubMed Scopus (49) Google Scholar, 12Torres J. Cooper C.E. Sharpe M. Wilson M.T. J. Bioenerg. Biomem. 1998; 30: 63-69Crossref PubMed Scopus (17) Google Scholar, 19Millar A.H. Day D.A. FEBS Lett. 1996; 398: 155-158Crossref PubMed Scopus (191) Google Scholar). Yu et al. (20Yu H. Sato E.F. Nagata K. Nishikawa M. Kashiba M. Arakawa T. Kobayashi K. Tamura T. Inoue M. FEBS Lett. 1997; 409: 161-165Crossref PubMed Scopus (56) Google Scholar) have shown that respiration of E. coli is also sensitive to NO in an O2-dependent fashion, with inhibition increasing as O2 tension decreases. Since whole cell respiration in the range of O2 tensions used in that study has contributions from the activity of both cytochromes bo′ and bd, each having a k m for O2 in the submicromolar range (43D'mello R. Hill S. Poole R.K. J. Bacteriol. 1995; 177: 867-870Crossref PubMed Google Scholar, 44D'mello R. Hill S. Poole R.K. Microbiology. 1996; 142: 755-763Crossref PubMed Scopus (182) Google Scholar), we tested separately the NO sensitivity of cytochrome bo′- and cytochrome bd-mediated respiration. A set of isogenic strains was used in which either cytochrome bd (strain AN2343, cydD) or bo′ (strain RKP4544,cyo) was absent. The phenotype of the strains was confirmed by room temperature absorption difference spectroscopy (cydD) or low temperature photodissociation spectroscopy (cyo); in the latter case absence of the characteristic 415/430 nm Soret bands of cytochrome o′ was confirmed (not shown). Strain AN2342 is a wild-type strain having both oxidases. Injection of NO solutions to respiring cell suspensions immediately inhibited oxygen uptake but the degree of inhibition was strongly dependent on the oxygen tension in the reaction chamber at the time of NO addition (Fig. 1 A). Thus, when 9 μm (final concentration) NO was added to a respiring cell suspension in which the O2 concentration was 33 μm (bottom trace), inhibition of respiration was virtually instantaneous and the residual respiration rate was only about 4% of the control value. After about 3 min, respiration resumed at a rate that slightly exceeded the initial rate and proceeded linearly until the oxygen in the chamber was depleted, reflecting the high oxygen affinities of the oxidases. However, when NO was added at successively higher dissolved O2 tensions, the extent of inhibition markedly decreased (e.g. to 25% of the control rate at 132 μm O2) and the period of inhibition was decreased. Qualitatively similar results were obtained with strain RKP4544 (cyo, not shown) and strain AN2343 (cydD, Fig. 1 B), although in both of these strains the period of respiratory inhibition at low O2tensions (25–33 μm) was always significantly greater than in the wild-type strain (Fig.2 A). Thus total respiratory capacity, rather than the properties of individual oxidases, may be the main determinant of NO inhibition of respiration at low oxygen tensions. At a fixed O2 concentration, the period of inhibition by NO was dose-dependent (shown for the wild-type strain only in Fig. 2 B). In view of the marked effects of O2 tension on the NO sensitivity of cell respiration, in subsequent experiments NO was added at predetermined O2 tensions, which are indicated on Figs.3, 4, and 5.Figure 3Sensitivity of respiratory oxygen consumption in an hmp mutant. Measurements of respiration of washed cell suspensions of E. coli strain RKP4545 (hmp) were made in an oxygen electrode apparatus. InA, a solution of NO was added at the arrows to give a final concentration in each case of 9 μm NO. The O2 concentration (μm) at the point of each addition is shown alongside each addition. The uninhibited respiration rate in each trace was 58 nmol of O2 consumed per min/mg of cell protein. The dashed lines in thetop trace in A shows the method of calculation of the period of inhibition. In B, lower concentrations of NO (2.5, 1.0, and 0.5 μm, top to bottom) were titrated to the cell suspension, and the initial inhibition is shown. The O2 concentration (μm) at each addition was 60 μm; traces are offset for clarity. C shows the inhibition of respiration, calculated from the steady state respiration rate immediately after addition of NO relative to the rate immediately before addition of the NO solution, as a function of NO concentration. The experiments were repeated at least three times with similar results.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 4Effects of NO on respiratory oxygen consumption of E. coli cells with altered Hmp expression. Measurements of respiration of washed cell suspensions were made in an oxygen electrode apparatus. In A, 9 μm NO was added at the arrows when the O2 concentration was 60–64 μm, as shown ontraces 1 (control, wild-type cells), 2 (wild-type cells grown with SNP), 3 (strain RSC521 containing pPL341), and 4 (strain RSC521 containing pPL341, grown with SNP).Numbers alongside the traces are respiration rates expressed as nanomole of O2 consumed per min/mg of cell protein. In the inset, 60 μm NO was added to the wild-type (trace 5) or RSC521 cells (Hmp-overproducing, trace 6) when the O2 concentration was 110 μm. In B, 9 μm NO was added at thearrows when the O2 concentration was 47–55 μm, as shown on traces 7 (strain RKP4545,hmp, but containing pPL341), 8 (strain RKP4545,hmp, no pPL341), and 9 (strain RKP4545,hmp, grown with SNP). The experiments were repeated at least 3 times with similar results. Inset C shows Western blots. Tracks are as follows: 1, purified Hmp; 2, strain RSC521 harboring pPL341; 3, wild-type strain AN2342;4, AN2342 grown with 100 μm SNP; 5,AN2342 grown with 100 μm GSNO; 6, hmp mutant RKP4545.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 5Sensitivity of respiratory oxygen consumption of E. coli cells to NO in the presence of cyanide. Measurements of respiration of washed cell suspensions ofE. coli wild-type strain AN2342 were made in an oxygen electrode apparatus. Trace 1 is a control in which NO but not cyanide was added. At dissolved oxygen tensions of 133–136 μm, cyanide was added to final concentrations of 100 (traces 2 and 3) or 200 μm(traces 4 and 5) where shown by the upper arrows. At dissolved oxygen tensions of 59–67 μm, a solution of NO was added to a final concentration of 9 μmwhere shown by the lower arrows. Numbersa" @default.
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• W1965481505 title "Flavohemoglobin Hmp Affords Inducible Protection for Escherichia coli Respiration, Catalyzed by Cytochromesbo′ or bd, from Nitric Oxide" @default.
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