Heterologous overexpression and purification of cytochrome c' from Rhodobacter capsulatus and a mutant (K42E) in the dimerization region. Mutation does not alter oligomerization but impacts the heme iron spin state and nitric oxide binding properties.
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 the heterologously expressed protein is assembled correctly, as judged by UV-vis absorption, EPR, and resonance Raman (RR) spectroscopy of the unligated protein as well as forms in which the heme is ligated by CO or NO. To probe the oligomerization state of RCCP and its potential influence on heme reactivity, we have compared the properties of wild-type RCCP with a mutant (K42E) that lacks a salt bridge at the subunit interface. Analytical ultracentrifugation indicates that wild-type and K42E proteins are 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 A distant), including perturbation of the ferric spin-state equilibrium and a change in the ferrous heme-nitrosyl complex from a six-coordinate/five-coordinate mixture to a predominantly five-coordinate heme-NO species. RR spectra indicate 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 a weaker Fe2+-His bond. Nevertheless, the altered reactivity of ferrous K42E with NO, together with its modified ferric spin state, shows that structural changes originating at the dimer interface can affect the properties of the heme center, raising the exciting possibility that intermolecular encounters at the protein surface might modulate the reactivity of cytochrome c' in vivo.
Department of Biology (Area 10), University of York, Heslington, York, YO10 5YW, United Kingdom.
Wilhelmina M
WM
Huston
Colin R
CR
Andrew
Amy E
AE
Servid
Alison L
AL
McKay
Andrew P
AP
Leech
Clive S
CS
Butler
James W B
JW
Moir
D002248:Carbon Monoxide / Q000737:chemistry; D045305:Cytochromes c' / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics*; D004578:Electron Spin Resonance Spectroscopy; D006418:Heme / Q000737:chemistry; D007501:Iron / Q000737:chemistry; D016297:Mutagenesis, Site-Directed; D009569:Nitric Oxide / Q000737:chemistry; D020836:Protein Structure, Quaternary; D016354:Rhodobacter capsulatus / Q000235:genetics / Q000378:metabolism*; D013056:Spectrophotometry, Ultraviolet; D013059:Spectrum Analysis, Raman
D045305:Cytochromes c'; D009569:Nitric Oxide; D006418:Heme; D002248:Carbon Monoxide; D007501:Iron