Denitrifying nitric oxide reductases (denNORs) are transmembrane proteins evolutionarily related to heme/copper terminal oxidases. They catalyze the two-electron reduction of NO to N2O, with wide consequences to the environment and human health. In addition to redox centers required for delivery of electron, the active sites of denNORs anchor a high-spin heme/nonheme (heme/FeB) binuclear center. The NO reduction is known to take place at the heme/FeB site, but the reaction mechanism and the coupling between electron and proton transfer are still unclear. To elucidate the mechanism of this reaction, we employ engineered myoglobin constructs (FeBMbs) that reproduce the structure of the diiron center of denNORs, and analyze their reaction with NO using FTIR, stopped-flow kinetics, and rapid freeze quench resonance Raman spectroscopy (RFQ-RR). Monitoring the formation of N2O by FTIR shows that in addition to the 3His/1Glu residues required to bind FeB, an additional Glu in the outer sphere of the binuclear site is needed for NOR activity. Stopped-flow and RFQ-RR data demonstrate that, with or without the outer-sphere Glu, a first NO binding to FeB(II) is kinetically favored and occurs prior to the binding of a second NO to the ferrous heme, leading to a [six-coordinate low-spin heme-nitrosyl/FeB-nitrosyl] transient complex. Without the outer-sphere Glu, this complex decays via dissociation of the heme proximal histidine to produce a [five-coordinate low-spin heme-nitrosyl/FeB�nitrosyl] dead-end complex. The added outer-sphere Glu decreases the rate of His dissociation and allows production of N2O. These results support a 'trans' mechanism for the NO reaction in FeBMbs  . Additional constructs are under investigation to better define the requirement for proton transfer in the NO reductase turnover. In parallel to our studies of FeBMb models, we investigate the reaction mechanism of purified cNOR from Paracoccus denitrificans. Kinetic data from stopped-flow spectroscopy will be discussed.

Kinetic and spectroscopic studies of NO reductase activity of heme-nonheme diiron centers

248th National Meeting (2014)

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