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 [Bioenergetics]

June 13th, 2014 by Singh, S., Thakur, N., Oliveira, A., Petruk, A. A., Hade, M. D., Sethi, D., Bidon-Chanal, A., Marti, M. A., Datta, H., Parkesh, R., Estrin, D. A., Luque, F. J., Dikshit, K. L.

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 micromole/min-1, which is nearly 3- and 5-fold faster than the ones reported for Vitreoscilla hemoglobin and myoglobin, respectively. Structural docking of the HbN with E. 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 electron coupling parameters calculated using the semiempirical pathway method amounts to an average of ca. 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 the FdR. The present study, thus, unravels 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.
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