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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.

DIR@IMTECH: CSIR-Institute of Microbial Technology

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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.
 
Creator Singh, Sandeep
Thakur, Naveen
Oliveira, Ana
Petruk, Ariel A
Hade, Mangesh Dattu
Sethi, Deepti
Bidon-Chanal, Axel
Martí, Marcelo A
Datta, Himani
Parkesh, Raman
Estrin, Dario A
Luque, F Javier
Dikshit, Kanak L
 
Subject QR Microbiology
 
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 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.
 
Publisher ASBMB
 
Date 2014-08-01
 
Type Article
PeerReviewed
 
Relation http://www.jbc.org/content/289/31/21573.long
http://crdd.osdd.net/open/1686/
 
Identifier Singh, Sandeep and Thakur, Naveen and Oliveira, Ana and Petruk, Ariel A and Hade, Mangesh Dattu and Sethi, Deepti and Bidon-Chanal, Axel and Martí, Marcelo A and Datta, Himani and Parkesh, Raman and Estrin, Dario A and Luque, F Javier and Dikshit, Kanak L (2014) 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. The Journal of biological chemistry, 289 (31). pp. 21573-83. ISSN 1083-351X