Converting a c-type to a b-type cytochrome: Met61 to His61 mutant of Pseudomonas cytochrome c-551.

Abstract

The gene nirM, coding for cytochrome c-551 in Pseudomonas stutzeri substrain ZoBell, was engineered to mutate Met61, the sixth ligand to the heme c, into His61, thereby converting the typical Met-His coordination of a c-type cytochrome into His-His, typical of b-type cytochromes. The mutant protein was expressed heterologously in Escherichia coli at levels 3-fold higher than in Pseudomonas and purified to homogeneity. The mutant retained low-spin visible spectral characteristics, indicating that the strong field ligand His 61 was coordinated to the iron. The physiochemical properties of the mutant were measured and compared to the wild-type properties. These included visible spectra, ligand binding reactions, stability to temperature and chemical denaturant, oxidation-reduction potentials, and electron-transfer kinetics to the physiological nitrite reductase of Pseudomonas. Despite a change in potential from the normal 260 mV to 55 mV, the mutant retained many of the properties of the c-551 family.