Ligand Binding to Truncated Hemoglobin N from Mycobacterium tuberculosis Is Strongly Modulated by the Interplay between the Distal Heme Pocket Residues and Internal Water

The survival of Mycobacterium tuberculosis requires detoxification of host ·NO. Oxygenated Mycobacterium tuberculosis truncated hemoglobin N catalyzes the rapid oxidation of nitric oxide to innocuous nitrate with a second-order rate constant (kNOD' ≈ 745 × 106m-1·s-1), which is ∼15-fold faster than the reaction of horse heart myoglobin. We ask what aspects of structure and/or dynamics give rise to this enhanced reactivity. A first step is to expose what controls ligand/substrate binding to the heme. We present evidence that the main barrier to ligand binding to deoxy-truncated hemoglobin N (deoxy-trHbN) is the displacement of a distal cavity water molecule, which is mainly stabilized by residue Tyr(B10) but not coordinated to the heme iron. As observed in the Tyr(B10)/Gln(E11) apolar mutants, once this kinetic barrier is lowered, CO and O2 binding is very rapid with rates approaching 1-2 × 109m-1·s-1. These large values almost certainly represent the upper limit for ligand binding to a heme protein and also indicate that the iron atom in trHbN is highly reactive. Kinetic measurements on the photoproduct of the ·NO derivative of met-trHbN, where both the ·NO and water can be directly followed, revealed that water rebinding is quite fast (∼1.49 × 108 s-1) and is responsible for the low geminate yield in trHbN. Molecular dynamics simulations, performed with trHbN and its distal mutants, indicated that in the absence of a distal water molecule, ligand access to the heme iron is not hindered. They also showed that a water molecule is stabilized next to the heme iron through hydrogen-bonding with Tyr(B10) and Gln(E11). The survival of Mycobacterium tuberculosis requires detoxification of host ·NO. Oxygenated Mycobacterium tuberculosis truncated hemoglobin N catalyzes the rapid oxidation of nitric oxide to innocuous nitrate with a second-order rate constant (kNOD' ≈ 745 × 106m-1·s-1), which is ∼15-fold faster than the reaction of horse heart myoglobin. We ask what aspects of structure and/or dynamics give rise to this enhanced reactivity. A first step is to expose what controls ligand/substrate binding to the heme. We present evidence that the main barrier to ligand binding to deoxy-truncated hemoglobin N (deoxy-trHbN) is the displacement of a distal cavity water molecule, which is mainly stabilized by residue Tyr(B10) but not coordinated to the heme iron. As observed in the Tyr(B10)/Gln(E11) apolar mutants, once this kinetic barrier is lowered, CO and O2 binding is very rapid with rates approaching 1-2 × 109m-1·s-1. These large values almost certainly represent the upper limit for ligand binding to a heme protein and also indicate that the iron atom in trHbN is highly reactive. Kinetic measurements on the photoproduct of the ·NO derivative of met-trHbN, where both the ·NO and water can be directly followed, revealed that water rebinding is quite fast (∼1.49 × 108 s-1) and is responsible for the low geminate yield in trHbN. Molecular dynamics simulations, performed with trHbN and its distal mutants, indicated that in the absence of a distal water molecule, ligand access to the heme iron is not hindered. They also showed that a water molecule is stabilized next to the heme iron through hydrogen-bonding with Tyr(B10) and Gln(E11). ·NO plays an important role in host defense against microbial pathogens by inhibiting or inactivating key enzymes such as the terminal respiratory oxidases (1.Brunori M. Trends Biochem. Sci. 2001; 26: 21-23Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar, 2.Brunori M. Giuffre A. Sarti P. Stubauer G. Wilson M.T. Cell. Mol. Life Sci. 1999; 56: 549-557Crossref PubMed Scopus (83) Google Scholar, 3.Stevanin T.M. 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Toxicol. 1997; 10: 1285-1292Crossref PubMed Scopus (566) Google Scholar, 9.Pfeiffer S. Gorren A.C.F. Schmidt K. Werner E. Hansert B. Bohle D.S. Mayer B. J. Biol. Chem. 1997; 272: 3465-3470Abstract Full Text Full Text PDF PubMed Scopus (304) Google Scholar). ·NO-metabolizing reactions are, thus, required to defend microbial pathogens against ·NO poisoning. In Mycobacterium tuberculosis the glbN gene encodes the truncated hemoglobin N (trHbN) 4The abbreviations used are: trHbN, M. tuberculosis truncated hemoglobin N; ·NO, nitric oxide; NOD, nitric oxide dioxygenase; Hb, hemoglobin; Mb, myoglobin; MD, molecular dynamics; DHP, distal heme pocket; 5C, 5 coordinated. 4The abbreviations used are: trHbN, M. tuberculosis truncated hemoglobin N; ·NO, nitric oxide; NOD, nitric oxide dioxygenase; Hb, hemoglobin; Mb, myoglobin; MD, molecular dynamics; DHP, distal heme pocket; 5C, 5 coordinated. (Fig. 1). Inactivation of glbN in Mycobacterium bovis bacillus Calmette-Guérin impairs the ability of stationary phase cells to protect aerobic respiration from nitric oxide (·NO) inhibition, suggesting that trHbN may protect M. tuberculosis from ·NO toxicity in vivo (10.Ouellet H. Ouellet Y. Richard C. Labarre M. Wittenberg B. Wittenberg J. Guertin M. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 5902-5907Crossref PubMed Scopus (237) Google Scholar). This functional assessment is supported by the observation that trHbN catalyzes the rapid oxidation of ·NO to nitrate (trHbN(Fe2+-O2)+·NO→trHbN(Fe3+)+NO3-), with a second-order rate constant kNOD' ≈ 745 × 106m-1·s-1 (23 °C) (10.Ouellet H. Ouellet Y. Richard C. Labarre M. Wittenberg B. Wittenberg J. Guertin M. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 5902-5907Crossref PubMed Scopus (237) Google Scholar). The nitric oxide dioxygenase (NOD) reaction catalyzed by trHbN is at least 15-fold faster (kNOD' ≈ 45 × 106m-1·s-1 at 23 °C) than the one recorded for horse heart myoglobin (Mb) and is almost as efficient as the diffusion-controlled reaction of ·NO with free O2.̲ A critical issue in this context is what aspects of structure and/or dynamics give rise to this enhanced reactivity. A first step is to expose what controls ligand/substrate binding to the heme. Once the ligand/substrate accesses the distal heme pocket (DHP), the issue of reactivity focuses on local factors such as iron reactivity and steric effects originating within the DHP. Inspection of Mb and trHbN structures shows that in Mb the imidazole ring of the proximal His is in an eclipsed orientation with respect to the pyrrole nitrogen atoms. 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Scopus Google Scholar). shows that of Tyr(B10) with or in an an of of both and both a steric with the of with or very on the binding for the the Tyr(B10) and is a that both and to the to the Tyr(B10) The as to what and/or responsible for effects on the As a first the and to ligand an In geminate which on the the of the the geminate on faster is to the responsible for the very large in rates from the the ligand is and within the local near the heme binding kinetic of the geminate and phase of CO to trHbN and distal on a The rebinding to trHbN of a This which with of CO is to phase The shows very fast the phase in to a in CO not indicating that this kinetic phase is a very fast phase The faster phase is with the that is a geminate reaction on both and to the CO The for the derivative of this showed and geminate on the not also on the yield at that for the as as the is a faster geminate phase for that the yield to the CO In to the CO derivative of the which an fast geminate with very large that is the for CO derivative of an or Mb low is almost geminate yield for trHbN. 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The in rates for the be to a in steric factors a in the of the to the in the that the of this steric The can for the observed one also of the of the by the against that from the observation that the of with or a on the on rates and on the geminate yield not for which is to that of the the binding rates for and very studies both at low and very not suggesting that the through a steric to the the and with respect to this steric A steric by the in and of the both the and with the in phase geminate and rates of geminate to the in a steric to a in the very that the not certainly on steric effects from the of the and with the absence of large ligand binding the proximal heme with trHbN U. Ouellet Y. Guertin M. J.M. J. Chem. PubMed Scopus Google factors that can to the of ligand binding in the of Mb and is to to the kinetic barrier for ligand binding Chem. 1994; Scopus Google Scholar, J. K. J. J. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, J. Biol. Chem. 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The yield and fast rates for the be to the very low of water within the DHP. this both kinetic measurements on the photoproduct of the ·NO derivative of met-trHbN, where both the ·NO and water can be directly followed, and to the of water within the of trHbN as a of and and studies indicated that Tyr(B10) and the coordinated water molecule in trHbN at 23 and Y. M. M. M. Guertin M. PubMed Scopus Google Scholar). the of apolar at Tyr(B10) and of heme with water coordinated to the iron atom Y. M. M. M. Guertin M. PubMed Scopus Google Scholar). 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Mb and where water not to geminate to of water the to ligand in the of trHbN the water on the of the geminate This observation that water access to the the heme iron on a that is faster than for This is with water stabilized within the protein at a near the heme iron or with water to the from the on a the of of the of deoxy-trHbN is not the present with that a water molecule, stabilized by Tyr(B10) and may be to the heme iron in the of a water molecule in the of deoxy-trHbN and its distal and to the role of Tyr(B10) and showed with protein The of the and the free water molecule shows the between atoms and the heme iron atom from the access to the heme iron atom used a of which to the of a water The in and as the of an the between atoms and the heme with and a water molecule in the water molecule fast the to a with and a water molecule in the The water molecule fast the to a in a and the iron with and a water molecule in the with and a water molecule in the DHP. in a produced for the and in both the water molecule stabilized by both the and Tyr(B10) As a the water molecule a main to the iron atom at a of of the water molecule this main to to the an the iron atom In the absence of a water molecule, the Tyr(B10) to the atom of Gln(E11). This the Tyr(B10) from the heme iron atom at a of This in of the In this the residue not ligand and the residue from the iron atom is at As a an the heme iron of the than in the the residue the water molecule from the heme at a of The water molecule a in the the residue showed and in and with one for trHbN. the heme iron atom observed than in trHbN The also than in trHbN by with simulations, kinetic showed a in both O2 and CO In the absence of a water molecule, the access to the heme iron atom reduced with respect to trHbN or and for the This is to the which to the heme iron as also observed in the structure of the derivative Y. M. M. M. Guertin M. PubMed Scopus Google Scholar). for the are, thus, with kinetic a water molecule is present in the DHP. the the water molecule a to that in trHbN and stabilized by a to the Tyr(B10) to the heme iron with respect to trHbN, for of the This is to the absence of by the residue water near the Tyr(B10) with and for the to of trHbN 1). In contrast, in the absence of a water molecule, cavity the heme iron atom in the with trHbN. In this the to as to the heme iron atom as the residue in the In trHbN, Tyr(B10) the at a from the heme iron atom through As a the the iron atom in the by than in trHbN in to kinetic in absence of a distal water molecule an in O2 and CO apolar a on the water molecule In simulations, the water molecule the protein through the These indicate that a water molecule is to within the of the apolar The of the revealed that apolar of the the by than with trHbN in the absence of a water molecule, than of the showed an the heme iron atom the in with the and which that iron of in this be by from the to the and to the present kinetic and indicate that the main barrier to ligand binding from and geminate phase to deoxy-trHbN is the displacement of a distal water molecule, which is mainly stabilized by the Tyr(B10) As observed for trHbN Tyr(B10)/Gln(E11) mutants, once this kinetic barrier is geminate yield is and ligand binding is very with rates approaching for diffusion-controlled a is supported by the observation that the rates for ·NO binding to heme-iron in the × with trHbN × 106m-1·s-1), as fast as CO and O2 binding to the form of the These large rates almost certainly represent the upper limit for ligand binding to a heme protein M. A. Ouellet Y. P. Guertin M. M. J. 2001; PubMed Scopus Google Scholar, M. E. P. A. M. J. Mol. Biol. PubMed Scopus Google Scholar) and also indicate that the heme iron in trHbN is highly reactive. rapid access to the is to the of the which may rapid and of the apolar the distal heme In the rapid of apolar ligand to the may be responsible for the efficient reaction catalyzed by trHbN × In to ligand water in the can in important and and with trHbN that of the rapid of the distal heme which of the bond and rapid of the nitrate from the A. A. J. Chem. PubMed Scopus Google Scholar). water in the the rapid of which is to an efficient detoxification and survival of the with that water to the distal heme at a rate of × 108 with and that water the distal heme pocket at a rate of × and × The large in the rates of water rebinding a barrier for water in trHbN, which can be in to the interactions of water with the distal Tyr(B10) and Gln(E11). The in binding rates of water between Mb and trHbN may also be to a faster access to the from the to the absence of a distal in trHbN J. K. J. J. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, M. A. Ouellet Y. P. Guertin M. M. J. 2001; PubMed Scopus Google Scholar, M. E. P. A. M. J. Mol. Biol. PubMed Scopus Google Scholar). The of the geminate studies with the water near the heme from the or the on an fast the to an important role for water in of ligand reactivity in trHbN. We to A. Wittenberg and B. Wittenberg from the of for is to

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