Mechanistic insight into the enzymatic reduction of truncated hemoglobin N of Mycobacterium tuberculosis: Role of the CD loop and Pre-A motif in electron cycling
Many pathogenic microorganisms have evolved hemoglobin-mediated nitric oxide (NO) detoxification mechanisms, where a globin domain in conjunction with a partner reductase catalyzes the conversion of toxic NO to innocuous nitrate. The truncated hemoglobin HbN of Mycobacterium tuberculosis displays a...
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todo:paper_00219258_v289_n31_p21573_Singh2023-10-03T14:23:22Z Mechanistic insight into the enzymatic reduction of truncated hemoglobin N of Mycobacterium tuberculosis: Role of the CD loop and Pre-A motif in electron cycling Singh, S. Thakur, N. Oliveira, A. Petruk, A.A. Hade, M.D. Sethi, D. Bidon-Chanal, A. Martí, M.A. Datta, H. Parkesh, R. Estrin, D.A. Luque, F.J. Dikshit, K.L. Detoxification Electron transitions Enzyme activity Escherichia coli Hemoglobin Nitric oxide Detoxification mechanism Enzymatic reduction Intermolecular interactions Mycobacterium tuberculosis Pathogenic microorganisms Site directed mutagenesis Truncated hemoglobins Vitreoscilla hemoglobin Reduction ferredoxin flavodoxin glycine heme hemoglobin n hemoglobin variant iron myoglobin reduced nicotinamide adenine dinucleotide truncated hemoglobin unclassified drug hemoglobin variant hemoglobins N primer DNA article catalysis controlled study electron transport Escherichia coli molecular docking Mycobacterium tuberculosis nonhuman priority journal protein motif protein protein interaction site directed mutagenesis Vitreoscilla chemistry electron electron transport enzymology genetics metabolism molecular dynamics Mycobacterium tuberculosis nucleotide sequence oxidation reduction reaction polymerase chain reaction Base Sequence DNA Primers Electron Transport Electrons Hemoglobins, Abnormal Molecular Dynamics Simulation Mutagenesis, Site-Directed Mycobacterium tuberculosis Oxidation-Reduction Polymerase Chain Reaction Many pathogenic microorganisms have evolved hemoglobin-mediated nitric oxide (NO) detoxification mechanisms, where a globin domain in conjunction with a partner reductase catalyzes the conversion of toxic NO to innocuous nitrate. The truncated hemoglobin HbN of Mycobacterium tuberculosis displays a potent NO dioxygenase activity despite lacking a reductase domain. The mechanism by which HbN recycles itself during NO dioxygenation and the reductase that participates in this process are currently unknown. This study demonstrates that the NADH-ferredoxin/flavodoxin system is a fairly efficient partner for electron transfer to HbN with an observed reduction rate of 6.2 μ-M/min -1 , which is nearly 3- and 5-fold faster than reported for Vitreoscilla hemoglobin and myoglobin, respectively. Structural docking of the HbN with Escherichia coli NADH-flavodoxin reductase (FdR) together with site-directed mutagenesis revealed that the CD loop of the HbN forms contacts with the reductase, and that Gly 48 may have a vital role. The donor to acceptor electron coupling parameters calculated using the semiempirical pathway method amounts to an average of about 6.4 10 -5 eV, which is lower than the value obtained for E. coli flavoHb (8.0 10 -4 eV), but still supports the feasibility of an efficient electron transfer. The deletion of Pre-A abrogated the heme iron reduction by FdR in the HbN, thus signifying its involvement during intermolecular interactions of the HbN and FdR. The present study, thus, unravels a novel role of the CD loop and Pre-A motif in assisting the interactions of the HbN with the reductase and the electron cycling, which may be vital for its NO-scavenging function. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc. Fil:Petruk, A.A. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:Martí, M.A. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:Estrin, D.A. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. JOUR info:eu-repo/semantics/openAccess http://creativecommons.org/licenses/by/2.5/ar http://hdl.handle.net/20.500.12110/paper_00219258_v289_n31_p21573_Singh |
| institution |
Universidad de Buenos Aires |
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I-28 |
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R-134 |
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Biblioteca Digital - Facultad de Ciencias Exactas y Naturales (UBA) |
| topic |
Detoxification Electron transitions Enzyme activity Escherichia coli Hemoglobin Nitric oxide Detoxification mechanism Enzymatic reduction Intermolecular interactions Mycobacterium tuberculosis Pathogenic microorganisms Site directed mutagenesis Truncated hemoglobins Vitreoscilla hemoglobin Reduction ferredoxin flavodoxin glycine heme hemoglobin n hemoglobin variant iron myoglobin reduced nicotinamide adenine dinucleotide truncated hemoglobin unclassified drug hemoglobin variant hemoglobins N primer DNA article catalysis controlled study electron transport Escherichia coli molecular docking Mycobacterium tuberculosis nonhuman priority journal protein motif protein protein interaction site directed mutagenesis Vitreoscilla chemistry electron electron transport enzymology genetics metabolism molecular dynamics Mycobacterium tuberculosis nucleotide sequence oxidation reduction reaction polymerase chain reaction Base Sequence DNA Primers Electron Transport Electrons Hemoglobins, Abnormal Molecular Dynamics Simulation Mutagenesis, Site-Directed Mycobacterium tuberculosis Oxidation-Reduction Polymerase Chain Reaction |
| spellingShingle |
Detoxification Electron transitions Enzyme activity Escherichia coli Hemoglobin Nitric oxide Detoxification mechanism Enzymatic reduction Intermolecular interactions Mycobacterium tuberculosis Pathogenic microorganisms Site directed mutagenesis Truncated hemoglobins Vitreoscilla hemoglobin Reduction ferredoxin flavodoxin glycine heme hemoglobin n hemoglobin variant iron myoglobin reduced nicotinamide adenine dinucleotide truncated hemoglobin unclassified drug hemoglobin variant hemoglobins N primer DNA article catalysis controlled study electron transport Escherichia coli molecular docking Mycobacterium tuberculosis nonhuman priority journal protein motif protein protein interaction site directed mutagenesis Vitreoscilla chemistry electron electron transport enzymology genetics metabolism molecular dynamics Mycobacterium tuberculosis nucleotide sequence oxidation reduction reaction polymerase chain reaction Base Sequence DNA Primers Electron Transport Electrons Hemoglobins, Abnormal Molecular Dynamics Simulation Mutagenesis, Site-Directed Mycobacterium tuberculosis Oxidation-Reduction Polymerase Chain Reaction Singh, S. Thakur, N. Oliveira, A. Petruk, A.A. Hade, M.D. Sethi, D. Bidon-Chanal, A. Martí, M.A. Datta, H. Parkesh, R. Estrin, D.A. Luque, F.J. Dikshit, K.L. Mechanistic insight into the enzymatic reduction of truncated hemoglobin N of Mycobacterium tuberculosis: Role of the CD loop and Pre-A motif in electron cycling |
| topic_facet |
Detoxification Electron transitions Enzyme activity Escherichia coli Hemoglobin Nitric oxide Detoxification mechanism Enzymatic reduction Intermolecular interactions Mycobacterium tuberculosis Pathogenic microorganisms Site directed mutagenesis Truncated hemoglobins Vitreoscilla hemoglobin Reduction ferredoxin flavodoxin glycine heme hemoglobin n hemoglobin variant iron myoglobin reduced nicotinamide adenine dinucleotide truncated hemoglobin unclassified drug hemoglobin variant hemoglobins N primer DNA article catalysis controlled study electron transport Escherichia coli molecular docking Mycobacterium tuberculosis nonhuman priority journal protein motif protein protein interaction site directed mutagenesis Vitreoscilla chemistry electron electron transport enzymology genetics metabolism molecular dynamics Mycobacterium tuberculosis nucleotide sequence oxidation reduction reaction polymerase chain reaction Base Sequence DNA Primers Electron Transport Electrons Hemoglobins, Abnormal Molecular Dynamics Simulation Mutagenesis, Site-Directed Mycobacterium tuberculosis Oxidation-Reduction Polymerase Chain Reaction |
| description |
Many pathogenic microorganisms have evolved hemoglobin-mediated nitric oxide (NO) detoxification mechanisms, where a globin domain in conjunction with a partner reductase catalyzes the conversion of toxic NO to innocuous nitrate. The truncated hemoglobin HbN of Mycobacterium tuberculosis displays a potent NO dioxygenase activity despite lacking a reductase domain. The mechanism by which HbN recycles itself during NO dioxygenation and the reductase that participates in this process are currently unknown. This study demonstrates that the NADH-ferredoxin/flavodoxin system is a fairly efficient partner for electron transfer to HbN with an observed reduction rate of 6.2 μ-M/min -1 , which is nearly 3- and 5-fold faster than reported for Vitreoscilla hemoglobin and myoglobin, respectively. Structural docking of the HbN with Escherichia coli NADH-flavodoxin reductase (FdR) together with site-directed mutagenesis revealed that the CD loop of the HbN forms contacts with the reductase, and that Gly 48 may have a vital role. The donor to acceptor electron coupling parameters calculated using the semiempirical pathway method amounts to an average of about 6.4 10 -5 eV, which is lower than the value obtained for E. coli flavoHb (8.0 10 -4 eV), but still supports the feasibility of an efficient electron transfer. The deletion of Pre-A abrogated the heme iron reduction by FdR in the HbN, thus signifying its involvement during intermolecular interactions of the HbN and FdR. The present study, thus, unravels a novel role of the CD loop and Pre-A motif in assisting the interactions of the HbN with the reductase and the electron cycling, which may be vital for its NO-scavenging function. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc. |
| format |
JOUR |
| author |
Singh, S. Thakur, N. Oliveira, A. Petruk, A.A. Hade, M.D. Sethi, D. Bidon-Chanal, A. Martí, M.A. Datta, H. Parkesh, R. Estrin, D.A. Luque, F.J. Dikshit, K.L. |
| author_facet |
Singh, S. Thakur, N. Oliveira, A. Petruk, A.A. Hade, M.D. Sethi, D. Bidon-Chanal, A. Martí, M.A. Datta, H. Parkesh, R. Estrin, D.A. Luque, F.J. Dikshit, K.L. |
| author_sort |
Singh, S. |
| title |
Mechanistic insight into the enzymatic reduction of truncated hemoglobin N of Mycobacterium tuberculosis: Role of the CD loop and Pre-A motif in electron cycling |
| title_short |
Mechanistic insight into the enzymatic reduction of truncated hemoglobin N of Mycobacterium tuberculosis: Role of the CD loop and Pre-A motif in electron cycling |
| title_full |
Mechanistic insight into the enzymatic reduction of truncated hemoglobin N of Mycobacterium tuberculosis: Role of the CD loop and Pre-A motif in electron cycling |
| title_fullStr |
Mechanistic insight into the enzymatic reduction of truncated hemoglobin N of Mycobacterium tuberculosis: Role of the CD loop and Pre-A motif in electron cycling |
| title_full_unstemmed |
Mechanistic insight into the enzymatic reduction of truncated hemoglobin N of Mycobacterium tuberculosis: Role of the CD loop and Pre-A motif in electron cycling |
| title_sort |
mechanistic insight into the enzymatic reduction of truncated hemoglobin n of mycobacterium tuberculosis: role of the cd loop and pre-a motif in electron cycling |
| url |
http://hdl.handle.net/20.500.12110/paper_00219258_v289_n31_p21573_Singh |
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