Mechanism of substrate inhibition in cytochrome-c dependent NO reductases from denitrifying bacteria (cNORs)

Steady-state kinetics of cytochrome-c dependent denitrifying NO reductases (cNORs) show evidence of substrate inhibition at NO concentrations higher than 10 mu M, but the mechanism of inhibition remains unclear. Here, we present low-temperature FTIR photolysis experiments carried out on the NO complex formed by addition of NO to the oxidized cNORs. A differential signal at 1261 cm(-1) that downshifts with (NO)-N-15 and (NO)-N-15-O-18 is assigned to a nu(NO2) from a bridging diiron-nitrito complex at the heme-nonheme diron site. Theoretical calculations re-produces observed frequencies and isotope shifts. Our experimental results confirm a prior theoretical study by Blomberg and Siegbahn [Blomberg, M. R., and Siegbahn, P. E. M. Biochemistry 2012, 51, 5173-5186] that proposed substrate inhibition through a radical combination reaction between the diferric mu-oxo group and an NO molecule to form a heme Fe(III)-nitrito-FeB(II) inhibitory complex. Stopped-flow experiments suggest that substrate inhibition also occurs after a half-reduction cycle, i.e. when fully-reduced cNOR reduces two NO molecules at the heme-nonheme diferrous active site cluster to produce one N2O molecule and the diferric cluster. These results support catalytic mechanisms that proceed through isomerization of a diferric-hyponitrite transient complex to produce a bridging diferric mu-oxo group and N2O without protonation of the putative hyponitrite intermediate.

Automated summary

In this study, low-temperature FTIR photolysis experiments were conducted to investigate the mechanism of substrate inhibition in cytochrome-c dependent denitrifying NO reductases (cNORs). The results confirmed that substrate inhibition is caused by a radical combination reaction between the diferric mu-oxo group and an NO molecule, forming a heme Fe(III)-nitrito-FeB(II) inhibitory complex.