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

S. SINGH; THAKUR, N.; A. OLIVEIRA; PETRUK, A.A.; HADE, M.D.; SETHI, D.; A. BIDON CHANAL; M. A. MARTI; DATTA, H.; PARKESH, R.; ESTRIN, D.A.; LUQUE, F.J.; DIKSHIT, K.L.

JOURNAL OF BIOLOGICAL CHEMISTRY

AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC

Año: 2014 vol. 289 p. 21573 - 21583

0021-9258

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 Gly48 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.