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.
abstract: 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 μM/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.
authors.affiliation: From the CSIR-Institute of Microbial Technology, Sector 39A, Chandigarh 160036, India.
authors.forename: Sandeep
authors.initials: S
authors.lastname: Singh
authors.affiliation: From the CSIR-Institute of Microbial Technology, Sector 39A, Chandigarh 160036, India.
authors.forename: Naveen
authors.initials: N
authors.lastname: Thakur
authors.affiliation: the Department de Fisicoquímica and Institut de Biomedicina (IBUB), Facultat de Farmàcia, Universitat de Barcelona, Campus de l'Alimentació Torribera, Santa Coloma de Gramenet, Spain, and.
authors.forename: Ana
authors.initials: A
authors.lastname: Oliveira
authors.affiliation: the Departamento de Química Inorgánica, Analítica, y Química Física/INQUIMAE CONICET, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Ciudad Universitaria, Pabellón 2, C1428EHA Buenos Aires, Argentina.
authors.forename: Ariel A
authors.initials: AA
authors.lastname: Petruk
authors.affiliation: From the CSIR-Institute of Microbial Technology, Sector 39A, Chandigarh 160036, India.
authors.forename: Mangesh Dattu
authors.initials: MD
authors.lastname: Hade
authors.affiliation: From the CSIR-Institute of Microbial Technology, Sector 39A, Chandigarh 160036, India.
authors.forename: Deepti
authors.initials: D
authors.lastname: Sethi
authors.affiliation: the Department de Fisicoquímica and Institut de Biomedicina (IBUB), Facultat de Farmàcia, Universitat de Barcelona, Campus de l'Alimentació Torribera, Santa Coloma de Gramenet, Spain, and.
authors.forename: Axel
authors.initials: A
authors.lastname: Bidon-Chanal
authors.affiliation: the Departamento de Química Inorgánica, Analítica, y Química Física/INQUIMAE CONICET, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Ciudad Universitaria, Pabellón 2, C1428EHA Buenos Aires, Argentina.
authors.forename: Marcelo A
authors.initials: MA
authors.lastname: Martí
authors.affiliation: From the CSIR-Institute of Microbial Technology, Sector 39A, Chandigarh 160036, India.
authors.forename: Himani
authors.initials: H
authors.lastname: Datta
authors.affiliation: From the CSIR-Institute of Microbial Technology, Sector 39A, Chandigarh 160036, India.
authors.forename: Raman
authors.initials: R
authors.lastname: Parkesh
authors.affiliation: the Departamento de Química Inorgánica, Analítica, y Química Física/INQUIMAE CONICET, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Ciudad Universitaria, Pabellón 2, C1428EHA Buenos Aires, Argentina.
authors.forename: Dario A
authors.initials: DA
authors.lastname: Estrin
authors.affiliation: the Department de Fisicoquímica and Institut de Biomedicina (IBUB), Facultat de Farmàcia, Universitat de Barcelona, Campus de l'Alimentació Torribera, Santa Coloma de Gramenet, Spain, and.
authors.forename: F Javier
authors.initials: FJ
authors.lastname: Luque
authors.affiliation: From the CSIR-Institute of Microbial Technology, Sector 39A, Chandigarh 160036, India, kanak@imtech.res.in.
authors.forename: Kanak L
authors.initials: KL
authors.lastname: Dikshit
mesh_terms: D001483:Base Sequence; D017931:DNA Primers; D004579:Electron Transport; D004583:Electrons; D006455:Hemoglobins, Abnormal / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D056004:Molecular Dynamics Simulation; D016297:Mutagenesis, Site-Directed; D009169:Mycobacterium tuberculosis / Q000737:chemistry / Q000201:enzymology / Q000378:metabolism*; D010084:Oxidation-Reduction; D016133:Polymerase Chain Reaction
chemical_list: D017931:DNA Primers; D006455:Hemoglobins, Abnormal; C031031:hemoglobins N
keywords: Electron Transfer; Hemoglobin; Hemoglobin Myoglobin; Microbial Pathogenesis; Molecular Biology; Mycobacterium Tuberculosis; Nitric Oxide