Truncated hemoglobin HbN protects Mycobacterium bovis from nitric oxide

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Abstract
Mycobacterium tuberculosis , the causative agent of human tuberculosis, and Mycobacterium bovis each express two genes, glbN and glbO , encoding distantly related truncated hemoglobins (trHbs), trHbN and trHbO, respectively. Here we report that disruption of M. bovis bacillus Calmette–Guérin glbN caused a dramatic reduction in the NO-consuming activity of stationary phase cells, and that activity could be restored fully by complementing knockout cells with glbN . Aerobic respiration of knockout cells was inhibited markedly by NO in comparison to that of wild-type cells, indicating a protective function for trHbN. TyrB10, which is highly conserved in trHbs and interacts with the bound oxygen, was found essential for NO consumption. Titration of oxygenated trHbN (trHbN⋅O 2 ) with NO resulted in stoichiometric oxidation of the protein with nitrate as the major product of the reaction. The second-order rate constant for the reaction between trHbN⋅O 2 and NO at 23°C was 745 μM −1 ⋅s −1 , demonstrating that trHbN detoxifies NO 20-fold more rapidly than myoglobin. These results establish a role for a trHb and demonstrate an NO-metabolizing activity in M. tuberculosis or M. bovis . trHbN thus might play an important role in persistence of mycobacterial infection by virtue of trHbN′s ability to detoxify NO.