Converting a c-type to a b-type cytochrome: Met61 to His61 mutant of Pseudomonas cytochrome c-551.
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.
Miller
G T
GT
Department of Chemistry, University of Alabama, Tuscaloosa, Alabama 35487-0336, USA.
Zhang
B
B
Hardman
J K
JK
Timkovich
R
R
D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D003573:Cytochrome b Group / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D003574:Cytochrome c Group / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D006639:Histidine / Q000235:genetics / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008715:Methionine / Q000235:genetics / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D011549:Pseudomonas / Q000201:enzymology* / Q000235:genetics; D013053:Spectrophotometry / Q000379:methods
D001426:Bacterial Proteins; D003573:Cytochrome b Group; D003574:Cytochrome c Group; D006639:Histidine; C017167:cytochrome C(551); D008715:Methionine