Heterologous Overexpression and Purification of Cytochrome c' from Rhodobacter capsulatus and a Mutant (K42E) in the Dimerization Region. Mutation Does Not Alter Oligomerization but Impa
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- 作者：Wilhelmina M. Huston ; Colin R. Andrew ; Amy E. Servid ; Alison L. McKay ; Andrew P. Leech ; Clive S. Butler ; and James W. B. Moir
- 刊名：Biochemistry
- 出版年：2006
- 出版时间：April 11, 2006
- 年：2006
- 卷：45
- 期：14
- 页码：4388 - 4395
- 全文大小：130K
- 年卷期：v.45,no.14(April 11, 2006)
- ISSN：1520-4995
文摘
Rhodobacter capsulatus cytochrome c' (RCCP) has been overexpressed in Escherichia coli,and its spectroscopic and ligand-binding properties have been investigated. It is concluded that theheterologously expressed protein is assembled correctly, as judged by UV-vis absorption, EPR, andresonance Raman (RR) spectroscopy of the unligated protein as well as forms in which the heme isligated by CO or NO. To probe the oligomerization state of RCCP and its potential influence on hemereactivity, we have compared the properties of wild-type RCCP with a mutant (K42E) that lacks a saltbridge at the subunit interface. Analytical ultracentrifugation indicates that wild-type and K42E proteinsare both monomeric in solution, contrary to the homodimeric structure of the crystalline state. Surprisingly,the K42E mutation produces a number of changes at the heme center (nearly 20 Å distant), includingperturbation of the ferric spin-state equilibrium and a change in the ferrous heme-nitrosyl complex froma six-coordinate/five-coordinate mixture to a predominantly five-coordinate heme-NO species. RR spectraindicate that ferrous K42E and wild-type RCCP both have relatively high Fe-His stretching frequencies,suggesting that the more favored five-coordinate heme-nitrosyl formation in K42E is not caused by aweaker Fe2+-His bond. Nevertheless, the altered reactivity of ferrous K42E with NO, together with itsmodified ferric spin state, shows that structural changes originating at the dimer interface can affect theproperties of the heme center, raising the exciting possibility that intermolecular encounters at the proteinsurface might modulate the reactivity of cytochrome c' in vivo.