Role of tryptophan 121 in the soluble CuA domain of cytochrome c oxidase: structure and electron transfer studies.
To investigate the contribution of tryptophan-121 (Trp121) residue to the structure and function of soluble CuA domain of cytochrome c oxidase, three mutant proteins, Trp121Tyr, Trp121Leu and Trp121-deleted mutant of the soluble domain of Paracoccus versutus cytochrome c oxidase, were constructed and expressed in Escherichia coli BL21 (DE3). Optical spectral studies showed that both the coordination structure of the CuA center and the secondary structure of the protein were changed significantly in the Leu substitution and deletion mutants of Trp121. Their electron transfer activity with cytochrome c was inhibited severely, as shown in stopped-flow kinetic studies. However, the CuA center can be reconstructed in the Trp121Tyr mutant although its stability decreases compared with the wild-type protein. This mutant keeps the same secondary structure as the wild-type protein, but can only transfer electrons with cytochrome c at a rate of one-seventh-fold. Based on the information on the structure, we also investigated and analyzed the possible factors that affect electron transfer. It appears that the aromatic ring, the size of the side chain and the hydrogen bonding ability of the Trp121 are crucial to the structure and function of the soluble CuA domain.
Song
Ai-Xin
AX
Chemical Biology Laboratory, Department of Chemistry, Fudan University, Shanghai 200433, China.
Li
Lian-Zhi
LZ
Yu
Ting
T
Chen
Shi-Ming
SM
Huang
Zhong-Xian
ZX
D000818:Animals; D003300:Copper / Q000737:chemistry*; D045304:Cytochromes c / Q000378:metabolism*; D004579:Electron Transport; D003576:Electron Transport Complex IV / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004583:Electrons; D004926:Escherichia coli / Q000235:genetics; D006736:Horses; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010230:Paracoccus / Q000201:enzymology*; D013329:Structure-Activity Relationship; D014364:Tryptophan / Q000737:chemistry* / Q000235:genetics
D003300:Copper; D014364:Tryptophan; D045304:Cytochromes c; D003576:Electron Transport Complex IV