Towards the Use of Time-Resolved X-ray Crystallography in Mechanistic Studies of Cytochrome c Nitrite Reductase from Shewanella Oneidensis

Abstract

A high-yield expression and purification of Shewanella oneidensis cytochrome c nitrite reductase (ccNiR), and its characterization by a variety of methods, notably Laue crystallography, is reported. A key component of the expression system is an artificial ccNiR gene in which the N-terminal signal peptide from the highly expressed S. oneidensis protein "Small Tetra-heme c" replaces the wild-type signal peptide. This gene, inserted into the plasmid pHSG298 and expressed in S. oneidensis TSP-C strain, generated approximately 20 mg crude ccNiR/L culture, compared with 0.5-1 mg/L for untransformed cells. Purified ccNiR exhibited nitrite and hydroxylamine reductase activities comparable to those of E. coli ccNiR. UV/Vis spectropotentiometric titrations identified five independent one-electron reduction processes. Global analysis of the spectropotentiometric data also allowed determination of the extinction coefficient spectra for the five reduced ccNiR species. Purified ccNiR yielded good quality crystals, with which the 2.59 Ä resolution structure was solved at 0&degC using the Laue diffraction method. The structure is similar to that of E. coli ccNiR, except in the region where each enzyme interacts with distinct physiological partners. Using the above-mentioned expression system, a mutant ccNiR bearing a surface cysteine was prepared and labeled with the photo-active [Ru(bpy)2(5-iodoacetamido-1,10-phenanthroline)]2+. In the presence of ferrocyanide the Ru-labeled ccNiR could be photo-reduced in less than 1&mus using a 5ns laser pulse. S. oneidensis ccNiR also oxidizes hydroxylamine, yielding nitrite as the sole nitrogenous product. UV/visible stopped-flow and rapid freeze-quench EPR data, along with product analysis, showed that the equilibrium between hydroxylamine and nitrite is fairly rapidly established in the presence of large initial concentrations of hydroxylamine, despite said equilibrium lying far to the left. By contrast reduction of hydroxylamine to ammonia did not occur, even though disproportionation of hydroxylamine to yield both nitrite and ammonia is strongly favored thermodynamically. This suggests a kinetic barrier to the ccNiR-catalyzed reduction of hydroxylamine to ammonia, which has important mechanistic implications. The availability of a large quantity of ccNiR, that yields crystals suitable for diffractometry using the Laue method, and that can be tagged with a photo-reductant, opens the door to future time-resolved X-ray crystallographic studies with this enzyme.