The distinct heme coordination environments and heme-binding stabilities of His39Ser and His39Cys mutants of cytochrome b5.
A gene mutant library containing 16 designed mutated genes at His39 of cytochrome b(5) has been constructed by using gene random mutagenesis. Two variants of cytochrome b(5), 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 b(5) >> His39Cys > His39Ser.
Chemical Biology Laboratory, Department of Chemistry and State Key Laboratory of Genetics, School of Life Science, Fudan University, Shanghai 200433, P.R. China.
Wen-Hu
WH
Wang
Jun-Xia
JX
Lu
Ping
P
Yao
Yi
Y
Xie
Zhong-Xian
ZX
Huang
D000818:Animals; D002417:Cattle; D003545:Cysteine / Q000235:genetics / Q000378:metabolism; D015786:Cytochromes b5 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006418:Heme / Q000737:chemistry* / Q000378:metabolism; D006639:Histidine / Q000235:genetics / Q000378:metabolism*; D013058:Mass Spectrometry; D009154:Mutation; D011489:Protein Denaturation; D012694:Serine / Q000235:genetics / Q000378:metabolism; D013696:Temperature
D006418:Heme; D012694:Serine; D006639:Histidine; D015786:Cytochromes b5; D003545:Cysteine