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Converting a c-type to a b-type cytochrome: Met61 to His61 mutant of Pseudomonas cytochrome c-551.
Miller, G T; Zhang, B; Hardman, J K; Timkovich, R.
Affiliation
  • Miller GT; Department of Chemistry, University of Alabama, Tuscaloosa, Alabama 35487-0336, USA.
Biochemistry ; 39(30): 9010-7, 2000 Aug 01.
Article in En | MEDLINE | ID: mdl-10913314
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.
Subject(s)
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Collection: 01-internacional Database: MEDLINE Main subject: Pseudomonas / Bacterial Proteins / Cytochrome b Group / Cytochrome c Group Language: En Journal: Biochemistry Year: 2000 Document type: Article Affiliation country: Estados Unidos
Search on Google
Collection: 01-internacional Database: MEDLINE Main subject: Pseudomonas / Bacterial Proteins / Cytochrome b Group / Cytochrome c Group Language: En Journal: Biochemistry Year: 2000 Document type: Article Affiliation country: Estados Unidos