Roles of the tyrosine diiron ligand of a mycobacterial hemerythrin-like protein on spectroscopic and redox properties, and catalase and nitric oxide reactivities

Tyrosine-ligated hemerythrin-like proteins (HLPs) are uniquely found in pathogenic mycobacteria. There is evidence that these HLPs have roles in virulence. To test the precise role of this tyrosine ligand, a Y54F HLP variant of Mycobacterium kansasii (Mka) was generated. There are substantial spectroscopic differences for the Y54F and the wild-type HLPs. First, the UV-visible absorbance spectrum of Y54F HLP lacked a 500-nm absorbance feature prevalent in the spectra of wild-type HLP and other Tyr-ligated diiron proteins. In addition, EPR spectroscopy showed that the Y54F HLP was isolated in a mixed-valent (FeII-FeIII) state, whereas wild-type HLP was isolated in the diferric state. In addition, the mixed-valent state of Y54F HLP exhibited a narrow, nearly axial EPR signal with g-values of 1.97, 1.93, and 1.89, a signal that is consistent with a µ-oxo bridged mixed-valent diiron site. Meanwhile, the wild-type counterpart exhibited a broad rhombic signal with simulated g-values of 1.99, 1.83, and 1.62, which is consistent with a µ-hydroxo bridged mixed-valent diiron site. Our results are consistent with Y54 coordination to the diiron site and indicate this ligand lowers the diiron [FeIII]2/FeIIFeIII redox couple and increases the basicity of the diiron solvent bridge. With regards to substrate reactivity, the Y54F mutation had no discernable effect on reductive nitrosylation reactivity, however, it precluded formation of the 520-nm intermediate observed when NO is reacted with wild-type Mka HLP. In addition, the Y54F mutation nearly eliminated catalase activity and greatly perturbed NO peroxidase activity. These results suggest that the Y54 ligand is critical for scavenging of H2O2 and NO, both of which are encountered during infection due to the host defense response.