The Truncated Oxygen-avid Hemoglobin from Bacillus subtilis

Abstract

The group II truncated hemoglobin from Bacillus subtilis has been cloned, expressed, purified, and characterized. B. subtilis truncated hemoglobin is a monomeric protein endowed with an unusually high oxygen affinity (in the nanomolar range) such that the apparent thermodynamic binding constant for O2 exceeds that for CO by 1 order of magnitude. The kinetic 2basis of the high oxygen affinity resides mainly in the very slow rate of ligand release. The extremely stable ferrous oxygenated adduct is resistant to oxidation, which can be achieved only with oxidant in large excess, e.g. ferricyanide in 50-fold molar excess. The three-dimensional crystal structure of the cyano-Met derivative was determined at 2.15 Å resolution. Although the overall fold resembles that of other truncated hemoglobins, the distal heme pocket displays a unique array of hydrophilic side chains in the topological positions that dominate the steric interaction with iron-bound ligands. In fact, the Tyr-B10, Thr-E7, and Gln-E11 oxygens on one side of the heme pocket and the Trp-G8 indole NE1 nitrogen on the other form a novel pattern of the “ligand-inclusive hydrogen bond network” described for mycobacterial HbO. On the proximal side, the histidine residue is in an unstrained conformation, and the iron-His bond is unusually short (1.91 Å). The group II truncated hemoglobin from Bacillus subtilis has been cloned, expressed, purified, and characterized. B. subtilis truncated hemoglobin is a monomeric protein endowed with an unusually high oxygen affinity (in the nanomolar range) such that the apparent thermodynamic binding constant for O2 exceeds that for CO by 1 order of magnitude. The kinetic 2basis of the high oxygen affinity resides mainly in the very slow rate of ligand release. The extremely stable ferrous oxygenated adduct is resistant to oxidation, which can be achieved only with oxidant in large excess, e.g. ferricyanide in 50-fold molar excess. The three-dimensional crystal structure of the cyano-Met derivative was determined at 2.15 Å resolution. Although the overall fold resembles that of other truncated hemoglobins, the distal heme pocket displays a unique array of hydrophilic side chains in the topological positions that dominate the steric interaction with iron-bound ligands. In fact, the Tyr-B10, Thr-E7, and Gln-E11 oxygens on one side of the heme pocket and the Trp-G8 indole NE1 nitrogen on the other form a novel pattern of the “ligand-inclusive hydrogen bond network” described for mycobacterial HbO. On the proximal side, the histidine residue is in an unstrained conformation, and the iron-His bond is unusually short (1.91 Å). Truncated hemoglobins (trHbs) 1The abbreviations used are: trHbs, truncated hemoglobins; PDB, protein data bank; Bs-trHb, B. subtilis trHb. are present in a wide variety of both Gram-positive and Gram-negative bacteria, as well as in some plants and fungi (1Wittenberg 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). TrHbs are 20–40 amino acids shorter than other globins, and the classical globin fold is not conserved as revealed by the crystal structures of the proteins from Chlamydomonas eugametus, Paramecium caudatum, and Mycobacterium tuberculosis (2Pesce A. Couture M. Dewilde S. Guertin M. Yamauchi K. Ascenzi P. Moens L. Bolognesi M. EMBO J. 2000; 19: 2424-2434Crossref PubMed Google Scholar, 3Milani M. Pesce A. Ouellet Y. Ascenzi P. Guertin M. Bolognesi M. EMBO J. 2001; 20: 3902-3909Crossref PubMed Scopus (193) Google Scholar, 4Milani M. Savard P.-Y. Ouellet H. Ascenzi P. Guertin M. Bolognesi M. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 5766-5771Crossref PubMed Scopus (106) Google Scholar). In contrast to vertebrate and other nonvertebrate hemoglobins, in which most heme pocket residues are conserved, bacterial hemoglobins and trHbs in particular are characterized by a remarkable variability in the nature of these residues, especially in the distal side of the heme pocket (1Wittenberg 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). Thus, whereas the proximal F8 histidine is invariant and the B9-B10 pair is commonly phenylalanine-tyrosine, other residues lining the heme pocket are not conserved. A phylogenetic classification has suggested the partition of trHbs in three groups that share less than 30% sequence similarity with each other, and the sequence identity within the same group may be greater than 80% (1Wittenberg 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 phylogenetic tree brings out distinct structural features of the active site that are characteristic of each group. In particular, group II and group III trHbs are characterized by the presence of a tryptophan residue at position G8, in the proximity of the heme group. Based on the crystal structure of M. tuberculosis HbO, the only available one for group II trHbs, such a large hydrophobic residue in the heme pocket is thought to hinder the escape of gaseous heme ligands and thereby contribute to the lower oxygen dissociation rate constants (around 10–3 s–1) observed in group II trHbs (5Watts R.A. Hunt P.W. Hvitved A.N. Hargrove M.S. Peacock W.J. Dennis E.S. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 10119-10124Crossref PubMed Scopus (174) Google Scholar, 6Ouellet H. Juszczak L. Dantsker D. Samuni U. Ouellet Y.H. Savard P.-Y. Wittenberg J.B. Wittenberg B.A. Friedman J.M. Guertin M. Biochemistry. 2003; 42: 5764-5774Crossref PubMed Scopus (98) Google Scholar) with respect to the group I proteins (10–1–10–2 s–1) (7Couture 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, 8Couture M. Das T.K. Lee H.C. Peisach J. Rousseau D.L. Wittenberg B.A. Wittenberg J.B. Guertin M. J. Biol. Chem. 1999; 274: 6898-6910Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar, 9Thorsteinsson M.V. Bevan D.R. M. Y. Hargrove M.S. Biochemistry. 1999; PubMed Scopus Google Scholar, T.K. Dewilde S. Wittenberg J.B. Wittenberg B.A. Yamauchi K. Moens L. Rousseau D.L. Biochemistry. 2000; PubMed Scopus Google Scholar, A.N. Hargrove M.S. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, M. Savard Ouellet H. Guertin M. Yeh S.R. Biochemistry. 2002; PubMed Scopus Google Scholar). to the of heme ligands is by hydrogen to Tyr-B10, a conserved residue in In some proteins a residue at position hydrogen to the ligand may residue that is on the side of the distal heme pocket to In HbO, has been to in ligand to the of an of residues in with the iron-bound ligand that has been a “ligand-inclusive hydrogen bond network” H. Juszczak L. Dantsker D. Samuni U. Ouellet Y.H. Savard P.-Y. Wittenberg J.B. Wittenberg B.A. Friedman J.M. Guertin M. Biochemistry. 2003; 42: 5764-5774Crossref PubMed Scopus (98) Google Scholar). The high oxygen affinity by most trHbs as oxygen (1Wittenberg 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, 6Ouellet H. Juszczak L. Dantsker D. Samuni U. Ouellet Y.H. Savard P.-Y. Wittenberg J.B. Wittenberg B.A. Friedman J.M. Guertin M. Biochemistry. 2003; 42: 5764-5774Crossref PubMed Scopus (98) Google Scholar). been e.g. trHbs been to be PubMed Scopus Google to as oxygen (1Wittenberg 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 to be in the to and (7Couture 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, H. Ouellet Y. M. Wittenberg B.A. Wittenberg J.B. Guertin M. Proc. Natl. Acad. Sci. U. S. A. 2002; PubMed Scopus Google to has been as bacteria, Mycobacterium and M. than one to In M. group I is the and is thought to be in the whereas has been to group II HbO, which is the Mycobacterium M. Savard P.-Y. Ouellet H. Ascenzi P. Guertin M. Bolognesi M. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 5766-5771Crossref PubMed Scopus (106) Google Scholar). features been with the of to the the M. Savard Ouellet H. Guertin M. Yeh S.R. Biochemistry. 2002; PubMed Scopus Google Scholar, J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar) and may to other In the large of trHbs in with a Mycobacterium is of for the of The for truncated hemoglobins been in of the Bacillus Bacillus Bacillus Bacillus and as well as Bacillus The proteins to group II trHbs, are to each other sequence and the same heme pocket in Bacillus been truncated hemoglobins with oxygen and in B. subtilis the of the oxygen as well as the heme share sequence similarity with the trHb. structural that residues within the heme pocket are not conserved, for the three proteins S. J. M. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). In the structure and ligand binding of Bacillus trHbs a the of the of these and from the other from and of B. subtilis of the from B. subtilis was The and The was with the with and and a with the and the with the in and heme and in the The in a of 1 and the was and the was on a with and with a was on a with The protein was on the and the protein determined and to to the from the protein sequence data The sequence of group II trHbs and M. tuberculosis HbO, and group I trHbs tuberculosis Chlamydomonas and Paramecium caudatum, three-dimensional structures been determined was with PubMed Scopus Google Scholar) and to the structural The sequence of B. subtilis was used to the sequence data for sequence the J. PubMed Scopus Google Scholar) and K. J. J. Y. M. 2003; PubMed Scopus Google Scholar). A sequence of the was and to the structural in The sequence not was used to sequence variability at and to the oxygenated derivative of cyano-Met Bs-trHb, by of molar to the was achieved at by the A of protein at for the cyano-Met was with an of the in a and at and in and very A data was at Å the on the at at a of The structure from a data the on the at at 2.15 Å resolution. The data at as with and with PubMed Scopus Google Scholar). The data that the crystal is with of a and the one The data an of and a The data is at 2.15 Å resolution. and structure of was by the A of M. tuberculosis as The and with the A. A. J. Scopus Google Scholar) in the of a of the and been out with the A. Biol. 1999; PubMed Scopus Google Scholar). has been the Scholar). The structure has been to an of and an of The residues 1 heme a and 1 The of the structure was by the R.A. J.M. J. Google Scholar). The overall as a of the of the is The of the residues in the most The most structural and are in data and of the data of of the data in of in in in a on a of the of of of the oxygenated derivative was determined on a with an at in The at an for protein in the to 1 at at a of with three The data with P. P. 2000; PubMed Scopus Google and the was to by S. in and of Scholar). at and at a of with with and with of both and at the of the to for the of was at on an array The derivative was by the oxygenated protein with 1 CO in a The protein was to a a of at oxygen The was the of at the of the an was of oxygenated at a protein of to a and with 1 The was the of at for In the was 1 and for the of the oxygen in a The protein was in The rate constant of oxygen was by a of with an of these the CO binding is and oxygen is the The in at to the of was The of the apparent rate of oxygen was by the oxygenated protein with of A was to and J. Biol. Chem. Full Text PDF PubMed Google Scholar) and to in the with at to the of the and at out by and with at in order to the of the In these the protein and nitrogen to with the The of CO binding to by a of with and CO at from to The in at was The CO binding of B. subtilis by The protein was in The protein was in a with a of 1 and was with CO at an of 1 The out at as an the from a with of and of The the to the of a a a as a The of at the of the derivative and a The CO dissociation by the adduct with in excess. The derivative was by a with 1 the the CO derivative was with a in a The at was a of out by oxygenated in to ferricyanide in excess. of the protein for in the presence of a of ferricyanide the oxidant and by a with at a with a and an The on the adduct and on the ferrous oxygenated derivative at and sequence of group II and group I trHbs, structural ligand binding been is in The three which the fold of trHbs, and residues in the heme pocket are In residue is as in bacterial hemoglobins, whereas residue is at with the other group II trHbs that a hydrophobic residue in position (1Wittenberg 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 and by residues, are both residues, and position the less conserved of the three-dimensional structure brings out and to M. tuberculosis HbO, the only group II hemoglobin crystal structure has been determined M. Savard P.-Y. Ouellet H. Ascenzi P. Guertin M. Bolognesi M. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 5766-5771Crossref PubMed Scopus (106) Google Scholar). The overall fold and the position of the heme and of the heme residues are in the Thus, three in to A and the and and the and and are in a The structural the proteins out by these a of Å The is to a in the of a the a characteristic of group II The the proteins are observed in the and the and In the is less than in in particular, the residues are in a in the which the is the and to be less than in an residue in In the and which is and the in HbO, in is shorter and of the of three residues, and of HbO, and to the of with a residue in distal heme pocket of is characterized by the presence of an array of residues the and The is to the heme and the is to the heme at a of The is by the which is the group to the nitrogen Å). is in the distal pocket and is to the heme with the indole NE1 at a of Å from the nitrogen the of the distal a residue a with the heme Å). is on the of the protein and may hinder to the distal position is by a residue in three groups of the heme is a by the and and the short The of the heme pocket in the proximal is by and and from position is by a residue The position and of the proximal histidine is of an unstrained that the heme of the Although the histidine to be a of the trHbs to U. Ouellet Y. Guertin M. Friedman J.M. Yeh S.R. J. Chem. PubMed Scopus Google the bond by is than in other hemoglobins as by the unusually short Å). is of that in the proximal pocket to be to a in a by the of the of and the heme The of the is by by the residues is to the oxygen of and that a with the oxygen of the heme D. In the and are conserved and an residue a is observed with a of the of the can be in by of an a J. H. Sci. PubMed Scopus Google The is by residues and on the and on the and and on the whereas the one is by residues and on the of the and by and on the The the is by and and of the and is in in the oxygenated of the extremely high oxygen can be achieved only of as is of CO to the was by The of ferrous and are in The of the ferrous oxygenated adduct was at with the of the derivative the same protein the presence of a of high and of a derivative of and Full of the oxygenated adduct to the of the a that the a of B. subtilis trHb. of the and ferrous oxygenated at K. was in at the presence of of in the of the oxygenated the of the ferrous oxygenated the adduct to be to the oxygenated adduct at in with of ferricyanide hydrogen in molar to slow heme as by the of the in the that of with in the not In ferricyanide in at molar a The ferricyanide by the at and a in the at and can be to a high and was by the presence of a of in at on the protein the protein an at than with the of an adduct and of on oxygenated an not of a monomeric The data and that the oxygenated derivative of B. subtilis is monomeric and not Thus, the data to a with of The to the be of a of the to an of be that the is stable the of the as the at at are on the derivative of the protein the of of to of oxygen in the and dissociation rate constants for oxygen and determined in and ligand The are in The rate of oxygen binding be by as ligand dissociation is in the on the oxygenated derivative the available Thus, the oxygen binding rate was from ligand binding and dissociation constants of B. subtilis and group II truncated subtilis was determined from O2 with CO in the presence of tuberculosis M. Das T.K. Lee H.C. Peisach J. Rousseau D.L. Wittenberg B.A. Wittenberg J.B. Guertin M. J. Biol. Chem. 1999; 274: 6898-6910Abstract Full Text Full Text PDF PubMed Scopus (110) Google tuberculosis M. Das T.K. Lee H.C. Peisach J. Rousseau D.L. Wittenberg B.A. Wittenberg J.B. Guertin M. J. Biol. Chem. 1999; 274: 6898-6910Abstract Full Text Full Text PDF PubMed Scopus (110) Google H. Juszczak L. Dantsker D. Samuni U. Ouellet Y.H. Savard P.-Y. Wittenberg J.B. Wittenberg B.A. Friedman J.M. Guertin M. Biochemistry. 2003; 42: 5764-5774Crossref PubMed Scopus (98) Google was determined from O2 with CO in the presence of in a The rate of oxygen was in oxygen dissociation with In vertebrate and the of the oxygenated protein with not on the observed rate is by the rate of oxygen from the heme In in the of the oxygenated derivative with is on the The observed at which of the was to a for The rate constant on the of than on with an overall rate of as from the of the of kinetic that the the which is in with as a at with the of with not oxygen by and be used to the of the rate constant of oxygen release. with out on in the of a rate of displays a and is at a rate of in with the by and J. Biol. Chem. Full Text PDF PubMed Google Scholar) at by a of a with an of in the presence of 1 these binding of CO to the heme is by the of CO with respect to and oxygen dissociation is and In these the was to be of the The CO rate was determined in The observed are slow and an apparent rate constant of an characteristic of group I (around s–1) and group II (around s–1) trHbs (5Watts R.A. Hunt P.W. Hvitved A.N. Hargrove M.S. Peacock W.J. Dennis E.S. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 10119-10124Crossref PubMed Scopus (174) Google Scholar, 6Ouellet H. Juszczak L. Dantsker D. Samuni U. Ouellet Y.H. Savard P.-Y. Wittenberg J.B. Wittenberg B.A. Friedman J.M. Guertin M. Biochemistry. 2003; 42: 5764-5774Crossref PubMed Scopus (98) Google Scholar, 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, 9Thorsteinsson M.V. Bevan D.R. M. Y. Hargrove M.S. Biochemistry. 1999; PubMed Scopus Google Scholar, T.K. Dewilde S. Wittenberg J.B. Wittenberg B.A. Yamauchi K. Moens L. Rousseau D.L. Biochemistry. 2000; PubMed Scopus Google Scholar, A.N. Hargrove M.S. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). on a and in that within the The rate of CO was in ligand by the derivative with oxygen and by the of the at The observed rate has a of In order to an of the rate constants for oxygen and CO and to the apparent rate constant of oxygen a of was out in the of In the ligand a of was to of oxygen a of was to of The can be PubMed Scopus (110) Google Scholar) as in In the presence of high ligand the of can be to be and the rate of to is as in A of the ligand in which the lower to the CO dissociation rate constant and the to the oxygen dissociation rate The in the to the data by the for the CO binding s–1) and dissociation s–1) rate constants as well as the the constants for O2 and CO to the The apparent rate of oxygen binding from the is very with a kinetic constant of is than the for other group II trHbs and is to the of group I The of the to a of 10–3 as a in the in with the rate of oxygen in the presence of CO and 10–3 The of the oxygen binding and dissociation constants an high for the The affinity of for CO is lower than that for with an constant of The three-dimensional structure and ligand binding of out features of protein to other group II trHbs and M. tuberculosis in particular, which to the by the available structural and data on The structure of the distal site is characterized by a of residues that are conserved in group II trHbs and by the presence of unique amino Thus, and are conserved at positions that dominate the steric with the iron-bound ligands. In position which is by an residue in and in A. a and of are by a and a as in A. trHb. The of these side chains the of a array the distal a in the of the ligand as by the presence of a hydrogen bond the oxygen and the nitrogen Å the nitrogen of and the group to the within the distal of the distal site with that of that the “ligand-inclusive of hydrogen is a of both and of amino side chains contribute to the of the the ligand in the The proximal site of is characterized by an that to form a bond with to other hemoglobins of Å in HbO, A residue at Å from the the only steric to the Thus, the in a with respect to the to the one and to an of the In mycobacterial and the heme group has a and the bond is unstrained as by U. Ouellet Y. Guertin M. Friedman J.M. Yeh S.R. J. Chem. PubMed Scopus Google Scholar). these to be to both group I and group II the of proximal bond of and in contrast with hemoglobins, in trHbs U. Ouellet Y. Guertin M. Friedman J.M. Yeh S.R. J. Chem. PubMed Scopus Google Scholar). the available and on trHbs is of an extremely active site with a in the distal and a proximal and distal heme pocket in B. subtilis and in M. tuberculosis distal heme distal heme distal heme distal heme in a The proximal pocket of displays a of the presence of a on one side by the heme and on the other by the of and the of which may of other to the proximal of the three-dimensional structure that the can be observed in protein residues and are conserved. The structural of the heme pocket in with the active site of brings out Thus, the heme (in the distal and the proximal in the observed in Bs-trHb, and the the proximal histidine and the residue the the and the and the of the residue are is that is endowed with In as to be hydrogen A structural of that a is as a monomeric in be out that data on trHbs are and with respect to both the used and to the The only data available to on the derivative of A. which is a (5Watts R.A. Hunt P.W. Hvitved A.N. Hargrove M.S. Peacock W.J. Dennis E.S. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 10119-10124Crossref PubMed Scopus (174) Google Scholar). at an of on M. tuberculosis the and are monomeric in the presence of and at lower H. Juszczak L. Dantsker D. Samuni U. Ouellet Y.H. Savard P.-Y. Wittenberg J.B. Wittenberg B.A. Friedman J.M. Guertin M. Biochemistry. 2003; 42: 5764-5774Crossref PubMed Scopus (98) Google Scholar). the cyano-Met derivative of M. tuberculosis as a of the high used in the and to the presence of that may and of M. Savard P.-Y. Ouellet H. Ascenzi P. Guertin M. Bolognesi M. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 5766-5771Crossref PubMed Scopus (106) Google Scholar). of the that only one of the of within the structure is to be stable in has a large and is by M. Savard P.-Y. Ouellet H. Ascenzi P. Guertin M. Bolognesi M. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 5766-5771Crossref PubMed Scopus (106) Google Scholar). of the residues at are in B. subtilis of are not conserved in one of the for the of the proteins in The ligand binding of a of the hydrogen bond that to of the heme ligands in group II In the by the hydrogen of the residue is by the of the and dissociation rate constants for O2 and CO in the of M. tuberculosis to the protein M. Savard P.-Y. Ouellet H. Ascenzi P. Guertin M. Bolognesi M. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 5766-5771Crossref PubMed Scopus (106) Google Scholar). A. and a residue in the position a of the presence of The of the residue in of may for the O2 and CO affinity of protein with respect to the other group II trHbs Although the structural of the slow rate of ligand in can be in the hydrogen of within the distal the very high oxygen rate as from the ligand be in is in with the heme by the unstrained proximal these features are to both and HbO, the of in the constant as the to oxygen CO within the is to that the high of the oxygenated adduct and to are to group II trHbs of the in with the heme The presence of a protein may for the of the protein in with and for the that the of oxygen with CO as on the presence of the of the apparent rate of oxygen on in the of CO that not an oxygen is to to the ferrous oxygenated protein 6Ouellet H. Juszczak L. Dantsker D. Samuni U. Ouellet Y.H. Savard P.-Y. Wittenberg J.B. Wittenberg B.A. Friedman J.M. Guertin M. Biochemistry. 2003; 42: 5764-5774Crossref PubMed Scopus (98) Google Scholar). may be that an by the is to form an within the protein that oxygen is to the heme The presence of a of in the of the oxygenated protein a that the of an within the of an on a protein In particular, the presence of both high and in the of the oxygenated derivative may be for by a of a ferrous adduct H. A. J. Chem. 2003; PubMed Scopus Google Scholar). In the of may be that the ferrous oxygenated is in with a oxygenated the ferrous a of the oxygen to I in such as heme and In order to the of a ferrous the that to an in the of of the and to the of I to be in In the of the oxygenated derivative out the that protein is in oxygen The and as a of in the of and of the for the of the and A. for the the of the and the data from the of is for the