Properties of a soluble domain of subunit C of a bacterial nitric oxide reductase.

Bacterial nitric oxide reductases are integral membrane proteins that catalyze the reduction of two molecules of nitric oxide to nitrous oxide and water. They are diverged members of the superfamily of heme/copper oxidases. The enzyme from Paracoccus denitrificans (NorBC) contains two subunits; NorB comprises the membrane-integrated active site, which harbors a heme iron/non-heme iron dinuclear center. NorC is a membrane-anchored c-type cytochrome and presumably the site of electron uptake. A DNA construct encoding the water-soluble domain of NorC (NorC(sol)) was coexpressed with the cytochrome c maturation genes in Escherichia coli. Using redox potentiometry, electronic absorption, circular dichroism (CD), magnetic CD (MCD), nuclear magnetic resonance, and electron paramagnetic resonance (EPR) spectroscopy the following observations were made: (i) NorC(sol) was folded into a alpha-helical structure. (ii) The low-spin heme iron was coordinated by histidine and methionine in both redox states. (iii) The midpoint redox potential of the NorC(sol) heme was 183 mV, much lower than the corresponding value of 275 mV in the NorBC complex. This points to an increased solvent exposure of the NorC(sol) heme compared to in the native NorBC complex and shows that the electronic properties of NorC are modulated by NorB in the complex. (iv) The EPR and MCD spectra of NorC(sol) were considered alongside the spectra of NorBC, which has helped to resolve the contribution that different redox centers make in the holo-enzyme complex.