Yeast Flavohemoglobin from Candida norvegensis

Abstract

Flavohemoglobin was isolated directly from the yeast Candida norvegensis and studied on its structural, spectral, and stability properties. In Candidaflavohemoglobin, the 155 N-terminal residues make a heme-containing domain, while the remaining 234 C-terminal residues serve as a FAD-containing reductase domain. A pair of His-95 and Gln-63 was assigned to the proximal and distal residues, respectively. In purification procedure FAD was partially dissociated on a Butyl-Toyopearl column, so that FAD-lacking flavohemoglobin was also obtainable. In this ferric species, the Soret and charge-transfer bands were all characteristic of a penta-coordinate form. Compared with the recombinant heme domain expressed in Escherichia coli, we have measured the autoxidation rate over a wide pH range. The resulting pH dependence curves were then analyzed in terms of a nucleophilic displacement mechanism. As a result, the heme domain was found to be extremely susceptible to autoxidation, its rate being more than 100 times higher than that of sperm whale MbO2. However, this inherently high oxidation rate was dramatically suppressed in Candida flavohemoglobin to an extent almost comparable to the stability of mammalian myoglobins. These new findings lead us to conclude that Candida flavohemoglobin, differently from bacterial flavohemoglobins, can serve as an oxygen storage protein in aerobic conditions. Flavohemoglobin was isolated directly from the yeast Candida norvegensis and studied on its structural, spectral, and stability properties. In Candidaflavohemoglobin, the 155 N-terminal residues make a heme-containing domain, while the remaining 234 C-terminal residues serve as a FAD-containing reductase domain. A pair of His-95 and Gln-63 was assigned to the proximal and distal residues, respectively. In purification procedure FAD was partially dissociated on a Butyl-Toyopearl column, so that FAD-lacking flavohemoglobin was also obtainable. In this ferric species, the Soret and charge-transfer bands were all characteristic of a penta-coordinate form. Compared with the recombinant heme domain expressed in Escherichia coli, we have measured the autoxidation rate over a wide pH range. The resulting pH dependence curves were then analyzed in terms of a nucleophilic displacement mechanism. As a result, the heme domain was found to be extremely susceptible to autoxidation, its rate being more than 100 times higher than that of sperm whale MbO2. However, this inherently high oxidation rate was dramatically suppressed in Candida flavohemoglobin to an extent almost comparable to the stability of mammalian myoglobins. These new findings lead us to conclude that Candida flavohemoglobin, differently from bacterial flavohemoglobins, can serve as an oxygen storage protein in aerobic conditions. nitric oxide 4-morpholineethanesulfonic acid 4-morpholinepropanesulfonic acid 3-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}-1-propanesulfonic acid 3-(cyclohexylamino)propanesulfonic acid circular dichroism hemoglobin oxyhemoglobin maltose-binding protein methemoglobin oxymyoglobin In studies of nonvertebrate myoglobins and hemoglobins, we have disclosed, for the first time, the very unique structures of a protozoan myoglobin from Paramecium caudatum (1Iwaasa H. Takagi T. Shikama K. J. Mol. Biol. 1989; 208: 355-358Crossref PubMed Scopus (58) Google Scholar) and a yeast flavohemoglobin from Candida norvegensis (2Iwaasa H. Takagi T. Shikama K. Zool. Sci. (Tokyo). 1991; 8: 1134Google Scholar, 3Iwaasa H. Takagi T. Shikama K. J. Mol. Biol. 1992; 227: 948-954Crossref PubMed Scopus (49) Google Scholar). Two such different types of globins are now established to occur widely in single-celled organisms. A first protein class, referred to as truncated hemoglobins, includes small hemoproteins characterized so far in the ciliated protozoa such as Paramecium caudatum,Tetrahymena pyriformis, and Tetrahymena thermophila (1Iwaasa H. Takagi T. Shikama K. J. Mol. Biol. 1989; 208: 355-358Crossref PubMed Scopus (58) Google Scholar, 4Iwaasa H. Takagi T. Shikama K. J. Biol. Chem. 1990; 265: 8603-8609Abstract Full Text PDF PubMed Google Scholar, 5Korenaga S. Igarashi J. Matsuoka A. Shikama K. Biochim. Biophys. Acta. 2000; 1543: 131-145Crossref PubMed Scopus (12) Google Scholar), in the unicellular green algaChlamydomonas eugametos (6Couture M. Chamberland H., St- Pierre B. Lafontaine J. Guertin M. Mol. Gen. Genet. 1994; 243: 185-197Crossref PubMed Scopus (85) Google Scholar), and in the eubacteria such asNostoc commune (7Potts M. Angeloni S.V. Ebel R.E. Bassam D. Science. 1992; 256: 1690-1692Crossref PubMed Scopus (98) Google Scholar) and Mycobacterium tuberculosis(8Couture M. Yeh S.-R. Wittenberg B.A. Wittenberg J.B. Ouellet Y. Rousseau D.L. Guertin M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 11223-11228Crossref PubMed Scopus (202) Google Scholar). The present state of this topic has been reviewed recently by Wittenberg et al. (9Wittenberg J.B. Bolognesi M. Wittenberg B.A. Guertin M. J. Biol. Chem. 2002; 277: 871-874Abstract Full Text Full Text PDF PubMed Scopus (331) Google Scholar). The second protein group, occurring in bacteria, yeast, and fungi, includes single-chain flavohemoglobins that consist of a N-terminal, heme-containing oxygen binding domain and a C-terminal, FAD-containing domain structurally related to NADH or NADPH reductase (10Vasudevan 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, 11Zhu H. Riggs A.F. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 5015-5019Crossref PubMed Scopus (137) Google Scholar, 12Ermler U. Siddiqui R.A. Cramm R. Friedrich B. EMBO J. 1995; 14: 6067-6077Crossref PubMed Scopus (162) Google Scholar, 13Oshino R. Oshino N. Chance B. Hagihara B. Eur. J. Biochem. 1973; 35: 23-33Crossref PubMed Scopus (49) Google Scholar, 14Oshino R. Asakura T. Takio K. Oshino N. Chance B. Hagihara B. Eur. J. Biochem. 1973; 39: 581-590Crossref PubMed Scopus (49) Google Scholar, 15Ilari A. Bonamore A. Farina A. Johnson K.A. Boffi A. J. Biol. Chem. 2002; 277: 23725-23732Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). Moreover, analysis of the microbial genomic sequences indicates that certain eubacteria, such as Bacillus subtilis and Staphylococcus aureus, contain both flavohemoglobin and truncated hemoglobin (16Pesce A. Couture M. Dewilde S. Guertin M. Yamauchi K. Ascenzi P. Moens L. Bolognesi M. EMBO J. 2000; 19: 2424-2434Crossref PubMed Google Scholar). As a matter of course, various biochemical functions other than the conventional oxygen transport or storage have been proposed so far for these ancient hemoproteins, but the precise in vivo activity is still unclear. However, recent studies strongly suggest that the most probable role of bacterial flavohemoglobins is in the nitric oxide (NO)1 detoxification. In bacteria, NO is a very toxic agent that can be produced by the metabolic reduction of inorganic nitrogen oxides. For theEscherichia coli flavohemoglobin (Hmp), Gardner et al. (17Gardner 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) reported the aerobic conversion of NO to nitrate (NO 3−). Under anoxic reducing conditions, this protein was found to bind NO reversibly and to reduce it to NO−, which resulted in the formation of nitrous oxide (N2O) via a dimeric reaction (18Kim S.O. Orii Y. Lloyd D. Hughes M.N. Poole R.K. FEBS Letter. 1999; 445: 389-394Crossref PubMed Scopus (142) Google Scholar). These defenses against the nitrosative stress would be analogous to the well characterized that aerobic from oxygen In yeast, flavohemoglobin is its being the conditions. In yeast Candida on the other Oshino et al. R. Oshino N. Chance B. Hagihara B. Eur. J. Biochem. 1973; 35: 23-33Crossref PubMed Scopus (49) Google Scholar) found that flavohemoglobin in a for and this we have studied in the present the structural, spectral, and stability of Candida flavohemoglobin with on the heme the role of yeast flavohemoglobin be have and stress the binding of oxygen to be the to functions in Butyl-Toyopearl was a of and were from and NADH were of and for and all other were of from were with and The of Candida norvegensis were for with in a of yeast and The of were from the of in an of and The in was and with of for and in the of as was from The were by for the were the was with and The was then to a Butyl-Toyopearl which been with in pH the was with a of the was with The hemoglobin was by on a with and The was the of the of yeast hemoglobin on a with a of from to pH these were as far as The of Candida flavohemoglobin was conversion an of was on the of the Chem. PubMed Google Scholar). For the FAD was also with an of in the of a FAD was as the of FAD to the heme In with FAD were as flavohemoglobin while with were referred to as the FAD-lacking flavohemoglobin were in a or were in a was with a dichroism were in a with a In the far were with hemoglobin in a and the of on the to the rate of autoxidation of oxyhemoglobin was measured in over a wide of pH and in the of A and was in a and in a The reaction was by the of and in the over were on the measured of For the state of the hemoglobin was to the ferric by the of The were and The pH of the reaction was and the with a pH In the of 155 N-terminal residues of Candida flavohemoglobin was to be expressed as a with binding protein The this heme domain was with the and a the The was the of to the domain The was coli and the were for in 100 and The was by the of to a of and was for with were with and The in were and in of The with was from the by and by on a column, and by the recombinant protein and was with for the heme-containing domain, the protein was on a which been with it from the heme domain was then as the with pH these were and the heme domain was nitrogen The for autoxidation was produced from the by in and in an The of the recombinant heme domain was conversion an of was on the of the Chem. PubMed Google Scholar). In we the acid a we isolated a protein from and found that this was of acid residues with an the and a the C-terminal (2Iwaasa H. Takagi T. Shikama K. Zool. Sci. (Tokyo). 1991; 8: 1134Google Scholar, 3Iwaasa H. Takagi T. Shikama K. J. Mol. Biol. 1992; 227: 948-954Crossref PubMed Scopus (49) Google Scholar). the time, and Riggs H. Riggs A.F. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 5015-5019Crossref PubMed Scopus (137) Google Scholar) also characterized a flavohemoglobin from yeast, S. its acid of residues from the was found Candida and flavohemoglobin has that the C-terminal is by the residues of so that we the of acid residues to be as in and was by analysis and by analysis as well Matsuoka A. Takagi T. Shikama K. Zool. 1995; Scholar). that the of 155 N-terminal residues has with that of a bacterial hemoglobin from S. H. PubMed Scopus Google Scholar). the N-terminal of Candida hemoglobin to be the heme-containing that can bind the other the of the remaining 234 C-terminal residues a but of with that of a methemoglobin reductase found in T. T. S. M. S. M. H. J. Biochem. (Tokyo). 96: PubMed Scopus Google Scholar, T. T. S. M. M. J. Biochem. (Tokyo). PubMed Scopus Google Scholar). is and is to have a FAD that can from In yeast flavohemoglobin was to the ferric it can be to the by the of NADH so as to be to bind oxygen In a yeast hemoglobin to its the have that of different As in the N-terminal domain is the heme-containing that the C-terminal domain serve as a FAD-containing reductase to the from its ferric to the in the of In to these we have circular dichroism for this protein in pH As a result, the was found to be with a of for sperm whale this was and the was to be or it of to the heme-containing domain a or we have an for the domain in coli In the of 155 N-terminal residues was to be expressed as a with such of Candida flavohemoglobin and its heme-containing domain in pH that the heme-containing domain has a of this being almost to that of sperm whale the of be as In the heme domain, of the residues can in a In His-95 is assigned to the proximal while Gln-63 is the distal As for the remaining FAD-containing domain, its was to be or this is in and that of the bacterial flavohemoglobin from was by al. U. Siddiqui R.A. Cramm R. Friedrich B. EMBO J. 1995; 14: 6067-6077Crossref PubMed Scopus (162) Google Scholar). In the Candida the probable were to be and on the with other H. Riggs A.F. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 5015-5019Crossref PubMed Scopus (137) Google U. Siddiqui R.A. Cramm R. Friedrich B. EMBO J. 1995; 14: 6067-6077Crossref PubMed Scopus (162) Google Scholar, Science. 1991; PubMed Scopus Google Scholar, 1991; PubMed Scopus Google Scholar). be that both and flavohemoglobins, a different pair of and is to the distal and proximal residues, U. Siddiqui R.A. Cramm R. Friedrich B. EMBO J. 1995; 14: 6067-6077Crossref PubMed Scopus (162) Google Scholar). The and Candida flavohemoglobins was In of these yeast hemoglobin is very to have been from the of a that a and the other a that has a reductase of the and genomic sequences that in is in with that of flavohemoglobin by and Riggs H. Riggs A.F. Proc. Natl. Acad. Sci. U. S. 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In to the was to the and stability have isolated oxyhemoglobin directly from the yeast to on a column, the hemoglobin was to a with pH The was then with a of the to and also a small of the ferric methemoglobin as the hemoglobin was with a of from to pH is such a to that Candida oxyhemoglobin was in The small the FAD-lacking with of FAD while the was for the FAD In this the flavohemoglobin to FAD-lacking species, from of the the of Candida flavohemoglobin both in the and in the In the flavohemoglobin, the was than the with an of this being in to sperm whale a of the time, a characteristic of binding of an FAD The also a from the In this a of FAD The was produced from by the of The was by a The of Candida flavohemoglobin are in of Candida in in in a new As a the of the FAD-lacking hemoglobin FAD are in both in the and in the ferric the or the protein but the FAD As a result, the is almost to that of the protein that was found However, the ferric was different from that of the In the Soret was and by a in to a of the In this we have proposed that myoglobins can be on the of the of the Soret of the to that of the that is to the higher than were for the of of than were for the for the myoglobins the distal K. Matsuoka A. J. Mol. Biol. 1989; PubMed Scopus Google Scholar). For such different of the Soret is as In the the of the ferric heme is by a which can be by a to the distal In of myoglobins the the by the of a K. Matsuoka A. Biol. 1994; PubMed Google Scholar). have this by the circular in terms of the or penta-coordinate for the ferric myoglobins A. N. Shikama K. Eur. J. Biochem. 1992; PubMed Scopus Google Scholar). In the FAD-lacking of Candida flavohemoglobin, a of was with the of a well of The of the was to these are characteristic of a penta-coordinate of ferric and are in with of Gln-63 to the distal from these that in the heme from of the FAD In the flavohemoglobin its high of was also found to to the was to indicates that the be produced from the by of the as is in the of myoglobin as the distal T. Y. Matsuoka A. M. K. Y. Shikama K. Biochim. Biophys. Acta. 1998; PubMed Scopus Google Scholar). the of flavohemoglobin that the is in the ferric U. Siddiqui R.A. Cramm R. Friedrich B. EMBO J. 1995; 14: 6067-6077Crossref PubMed Scopus (162) Google A. D. M. U. Eur. J. Biochem. 1999; PubMed Scopus Google Scholar). In of Candida flavohemoglobin, it is a matter of that the was a very by a reduction with NADH present in the As for the of the FAD-lacking species, it is to a of FAD in the a of FAD was the hemoglobin was to a Butyl-Toyopearl that of the FAD from Candida flavohemoglobin the of such a on In Candida flavohemoglobin, a was reported by Oshino et al. R. Asakura T. Takio K. Oshino N. Chance B. Hagihara B. Eur. J. Biochem. 1973; 39: 581-590Crossref PubMed Scopus (49) Google Scholar), but the stability of the is to be of to the role of Candida flavohemoglobin on the of its unique it is in the that myoglobin or hemoglobin can bind oxygen Under conditions, the is to be to the ferric with of the as in the rate a pH T. Shikama K. J. Biochem. (Tokyo). PubMed Scopus Google Scholar). the rate of the autoxidation is as was by a of as the of to that can be by the of the in the such to the autoxidation rate of Candida oxyhemoglobin with of sperm and the in pH and in the of the of the rate was as for sperm whale for Paramecium for Candidaflavohemoglobin, and for the FAD-lacking the is for the flavohemoglobin, the the protein but the FAD is that Candida flavohemoglobin is to the ferric its rate being than that of the protozoan myoglobin from P. caudatum but times than that of sperm whale is also that a of FAD Candida flavohemoglobin extremely susceptible to autoxidation with a of high oxidation rate was with a of in the sperm whale was with a of the produced bind the rate can be as a for the stability of the K. Chem. 1998; PubMed Scopus Google Scholar). the of are against the pH of the a of the stability of oxyhemoglobin can be such flavohemoglobin and its FAD-lacking In the flavohemoglobin, the rate of autoxidation with but so than in sperm whale MbO2. In the a was found for the of as be in In Candida flavohemoglobin, its was than a the extremely pH is a that Candida flavohemoglobin contain such as the that can a role in the autoxidation reaction of most mammalian that have the distal In Candida flavohemoglobin, a rate pH and a the higher pH the its rate to a of the FAD-lacking an rate to from of the FAD from the reductase domain. In the pH which of the FAD was dissociated flavohemoglobin was on the and of Candida for the stability of the recombinant heme domain of Candida flavohemoglobin and the sperm whale myoglobin in The was by a to the over the of the pH studied on for sperm whale and for respectively. of sperm whale myoglobin are from Shikama and Matsuoka K. Matsuoka A. 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Riggs A.F. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 5015-5019Crossref PubMed Scopus (137) Google Scholar) that the has been for as as the of of and all yeast flavohemoglobin, differently from bacterial flavohemoglobins, can serve as an oxygen storage protein in aerobic functions the autoxidation of are to with such as