Converting a c-type to a b-type Cytochrome: Met61 to His61 Mutant of Pseudomonas Cytochrome c-551

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• 作者：Gregory T. Miller ; Balli Zhang ; John K. Hardman ; and Russell Timkovich
• 刊名：Biochemistry
• 出版年：2000
• 出版时间：August 1, 2000
• 年：2000
• 卷：39
• 期：30
• 页码：9010 - 9017
• 全文大小：85K
• 年卷期：v.39,no.30(August 1, 2000)
• ISSN：1520-4995

文摘

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