The hemerythrin-like diiron protein from Mycobacterium kansasii is a nitric oxide peroxidase

Abstract

The hemerythrin-like protein from Mycobacterium kansasii (Mka HLP) is a member of a distinct class of oxo-bridged diiron proteins that are found only in mycobacterial species that cause respiratory disorders in humans. Because it had been shown to exhibit weak catalase activity and a change in absorbance on exposure to nitric oxide (NO), the reactivity of Mka HLP toward NO was examined under a variety of conditions. Under anaerobic conditions, we found that NO was converted to nitrite (NO₂^-) via an intermediate, which absorbed light at 520 nm. Under aerobic conditions NO was converted to nitrate (NO₃^-). In each of these two cases, the maximum amount of nitrite or nitrate formed was at best stoichiometric with the concentration of Mka HLP. When incubated with NO and H₂O₂, we observed NO peroxidase activity yielding nitrite and water as reaction products. Steady-state kinetic analysis of NO consumption during this reaction yielded a K_m for NO of 0.44 μM and a k_cat/K_m of 2.3 × 10⁵ M^-1s^-1. This high affinity for NO is consistent with a physiological role for Mka HLP in deterring nitrosative stress. This is the first example of a peroxidase that uses an oxo-bridged diiron center and a rare example of a peroxidase utilizing NO as an electron donor and cosubstrate. This activity provides a mechanism by which the infectious Mycobacterium may combat against the cocktail of NO and superoxide (O₂^•-) generated by macrophages to defend against bacteria, as well as to produce NO₂^- to adapt to hypoxic conditions.

Keywords: catalase; mycobacteria; nitric oxide dioxygenase; nitric oxide oxidase; nitrosylation; nonheme iron; tuberculosis.

Figure 1

Structure of the Mka HLP.A, cartoon of the overall structure with the irons and bridging oxygen indicated. B, the oxo-bridged diiron site and amino acid ligands. HLP, hemerythrin-like protein; Mka, Mycobacterium kansasii.

Figure 2

Spectroscopic features of the as-isolated and dithionite-reduced Mka HLP.A, absorbance spectra of the Mka HLP. The spectra of the protein were recorded as-isolated (red) and after addition of dithionite (blue). B, EPR spectra of the Mka HLP. The spectra of the protein were recorded as-isolated (red) and after addition of dithionite (blue). The background spectrum of the cavity is black. Spectra were collected at 1 mW and 17 K. The inset shows a spectrum of the dithionite-reduced sample after subtraction of the cavity signal with the g values indicated. These spectra were collected at 5 mW and 17 K. HLP, hemerythrin-like protein; Mka, Mycobacterium kansasii.

Figure 3

Changes in the absorbance spectrum of the Mka HLP after addition of NO gas under anaerobic conditions. The spectra were recorded before NO addition (red), after mixing with NO gas (green) and 1500 s after NO addition (blue). Samples contained 60 μM HLP. HLP, hemerythrin-like protein; Mka, Mycobacterium kansasii.

Figure 4

EPR spectra of samples resulting from the anaerobic reaction of the Mka HLP with NO. Samples contained 200 μM HLP. Reaction was initiated with addition of NO gas to form the 520-nm intermediate (green) and incubated at room temperature under anaerobic conditions for 30 min to form the anaerobic product (blue). The background signal from the cavity is gray. Spectra were collected at 15 to 17 K, at 10 G modulation amplitude, and 1 mW microwave power. HLP, hemerythrin-like protein; Mka, Mycobacterium kansasii.

Figure 5

Scheme of the anaerobic reaction of the Mka HLP with NO. The bridging oxygen that interacts with the two irons is not shown for simplicity. HLP, hemerythrin-like protein; Mka, Mycobacterium kansasii.

Figure 6

Changes in the absorbance spectrum on aerobic addition of increasing amounts of NO. The initial spectrum before NO additions is red and the final spectrum after NO additions is blue. The spectra resulting from incremental additions of NO are black and the direction of the spectral changes are indicated by arrows. NO, nitric oxide.

Figure 7

Mka HLP and H₂O₂-dependent NO consumption. Representative traces of NO consumption as monitored by an NO electrode. Samples were in degassed 50 mM phosphate, pH 7.5 at room temperature and contained the concentration of H₂O₂ indicated in the legend. The dashed gray line indicates when 1 μM HLP was added to each sample. HLP, hemerythrin-like protein; Mka, Mycobacterium kansasii; NO, nitric oxide.

Figure 8

Steady-state kinetic analysis of NO consumption during the NO peroxidase reaction catalyzed by the Mka HLP. The concentration of NO was monitored using an NO electrode. NO was varied in the presence of 100 nM Mka HLP and 100 mM H₂O₂ in 50 mM potassium phosphate, pH 7.5, at 20 °C, under anaerobic conditions. Points are the average of three replicates. The line is a fit of the data to Equation 2 with an R² of 0.98. HLP, hemerythrin-like protein; Mka, Mycobacterium kansasii; NO, nitric oxide.

Figure 9

Proposed mechanisms for the NO peroxidase reaction that is catalyzed by the Mka HLP. Mechanism A utilizes only the oxo-bridged diiron site. Mechanism B also utilizes the Tyr residue that provides a ligand for iron. The bridging O is not shown for simplicity. HLP, hemerythrin-like protein; Mka, Mycobacterium kansasii; NO, nitric oxide.