Cyanide replaces substrate in obligate-ordered addition of nitric oxide to the non-heme mononuclear iron AvMDO active site

Thiol dioxygenases are a subset of non-heme mononuclear iron oxygenases that catalyze the O 2 -dependent oxidation of thiol-bearing substrates to yield sulfinic acid products. Cysteine dioxygenase (CDO) and 3-mercaptopropionic acid ( 3MPA ) dioxygenase (MDO) are the most extensively characterized members of this enzyme family. As with many non-heme mononuclear iron oxidase/oxygenases, CDO and MDO exhibit an obligate-ordered addition of organic substrate before dioxygen. As this substrate-gated O 2 -reactivity extends to the oxygen-surrogate, nitric oxide (NO), EPR spectroscopy has long been used to interrogate the [substrate:NO:enzyme] ternary complex. In principle, these studies can be extrapolated to provide information about transient iron-oxo intermediates produced during catalytic turnover with dioxygen. In this work, we demonstrate that cyanide mimics the native thiol-substrate in ordered-addition experiments with MDO cloned from Azotobacter vinelandii ( Av MDO). Following treatment of the catalytically active Fe(II)- Av MDO with excess cyanide, addition of NO yields a low-spin ( S = 1/2) (CN/NO)-Fe-complex. Continuous wave and pulsed X-band EPR characterization of this complex produced in wild-type and H157N variant Av MDO reveal multiple nuclear hyperfine features diagnostic of interactions within the first- and outer-coordination sphere of the enzymatic Fe-site. Spectroscopically validated computational models indicate simultaneous coordination of two cyanide ligands replaces the bidentate (thiol and carboxylate) coordination of 3MPA allowing for NO-binding at the catalytically relevant O 2 -binding site. This promiscuous substrate-gated reactivity of Av MDO with NO provides an instructive counterpoint to the high substrate-specificity exhibited by mammalian CDO for l -cysteine. Graphical abstract