Spectroscopic Characterization of a Diferric Mycobacterial Hemerythrin-Like Protein with Unprecedented Reactivity toward Nitric Oxide.

Hemerythrin-like proteins (HLPs) are broadly distributed across taxonomic groups and appear to play highly diverse functional roles in prokaryotes. Mycobacterial HLPs contribute to the survival of these pathogenic bacteria in mammalian macrophages, but their modes of action remain unclear. A recent crystallographic characterization of HLP (Mka-HLP) revealed the unexpected presence of a tyrosine sidechain (Tyr54) near the coordination sphere of one of the two iron centers. Here, we show that Tyr54 is a true ligand to the Fe2(III) ion which, in conjunction with the presence of a μ-oxo group bridging the two iron(III), brings unique reactivity toward nitric oxide (NO). Monitoring the titration of -HLP with NO by Fourier-transform infrared and electron paramagnetic resonance spectroscopies shows that both diferric and diferrous forms of -HLP accumulate an uncoupled high-spin and low-spin {FeNO} pair. We assign the reactivity of the diferric protein to an initial radical reaction between NO and the μ-oxo bridge to form nitrite and a mixed-valent diiron center that can react further with NO. Amperometric measurements of NO consumption by -HLP confirm that this reactivity can proceed at low micromolar concentrations of NO, before additional NO consumption, supporting a NO scavenging role for mycobacterial HLPs.