A single outer-sphere amino-acid substitution turns on the NO reactivity of a hemerythrin-like protein

Mycobacterial hemerythrin-like proteins (HLPs) are important for the survival of pathogens in macrophages. Their molecular mechanisms of function remain poorly defined but recent studies point to their possible role in nitric oxide (NO) scavenging. Unlike any nonheme diiron protein studied so far, the diferric HLP from <i>Mycobacterium kansasii</i> (<i>Mka</i>-HLP) reacts with NO in a multistep fashion to consume four NO molecules per diiron center. HLPs are largely conserved across mycobacteria and we argued that comparative studies of distant orthologs may illuminate the role of the protein scaffold in this reactivity and yield intermediates with properties more favorable for detailed spectroscopic characterization. Herein, we show that HLP from <i>Azotobacter vinelandii</i> (<i>Avi</i>-HLP) requires a single T47F point mutation in the outer sphere of its diferric center to adopt a bridging μ-oxo diferric structure as in <i>Mka</i>-HLP and makes it reactive toward NO. Radical combination of NO with the μ-oxo bridge yields nitrite and a mixed valent Fe(iii)Fe(ii) cluster that further react with NO to produce a stable magnetically coupled Fe(iii){FeNO}⁷ cluster. We report characterization of this stable cluster by electronic absorption, EPR, FTIR and resonance Raman spectroscopies and suggest ways Phe 46 (<i>Mka</i> numbering) might control the Fe(iii) reduction potential and the NO reactivity of HLPs.