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.
Highlights
The HbN of Mycobacterium tuberculosis carries a potent nitric-oxide dioxygenase activity despite lacking a reductase domain
Interactions of Mycobacterium tuberculosis (Mtb) HbN with Native and Heterologous Flavoreductases—Because NADP-ferredoxin/flavodoxin reductase systems are known to reduce met-Hb into the ferrous state [43] and similar reductase modules are found naturally fused with globins in two-domain flavoHbs, a series of flavoreductase systems were selected for testing the enzymatic reduction of MtbHbN
The missing link in understanding the NO dioxygenase (NOD) function of the MtbHbN, which is integral to its uninterrupted NOD activity, is the molecular mechanism by which HbN associates with a reductase partner to replenish itself from a ferric to ferrous state for the cycle of NOD activity
Summary
The HbN of Mycobacterium tuberculosis carries a potent nitric-oxide dioxygenase activity despite lacking a reductase domain. The truncated hemoglobin HbN of Mycobacterium tuberculosis displays a potent NO dioxygenase activity despite lacking a reductase domain. Raman spectroscopy revealed that heme iron coordination in MtbHbN is well suited for performing O2/NO chemistry for NO dioxygenation [7], which is modulated by two unusual structural features of the HbN: a 12-residue long highly flexible N-terminal Pre-A region that is required for the optimal NOD activity [8], and a protein tunnel system composed of short and long branches [9] that facilitates ligand entry to the distal heme site [9, 10]. Bacterial and yeast flavoHbs, acting as NO-dioxygenase enzymes, are endowed with heme and flavin binding domains
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