Protein Engineering vol. 16 no. 12 pp. 1047-1054, 2003
© 2003 Oxford University Press
The distinct heme coordination environments and heme-binding stabilities of His39Ser and His39Cys mutants of cytochrome b5
1Chemical Biology Laboratory, Department of Chemistry and 2State Key Laboratory of Genetics, School of Life Science, Fudan University, Shanghai 200433, P.R. China
3 To whom correspondence should be addressed. e-mail: zxhuang{at}fudan.edu.cn
A gene mutant library containing 16 designed mutated genes at His39 of cytochrome b5 has been constructed by using gene random mutagenesis. Two variants of cytochrome b5, His39Ser and His39Cys mutant proteins, have been obtained. Protein characterizations and reactions were performed showing that these two mutants have distinct heme coordination environments: ferric His39Ser mutant is a high-spin species whose heme is coordinated by proximal His63 and likely a water molecule in the distal pocket, while ferrous His39Ser mutant has a low-spin heme coordinated by His63 and Ser39; on the other hand, the ferric His39Cys mutant is a low-spin species with His63 and Cys39 acting as two axial ligands of the heme, the ferrous His39Cys mutant is at high-spin state with the only heme ligand of His63. These two mutants were also found to have quite lower heme-binding stabilities. The order of stabilities of ferric proteins is: wild-type cytochrome b5 >> His39Cys > His39Ser.
Received April 11, 2003; revised October 13, 2003; accepted October 23, 2003
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