Ligand-induced monomerization of Allochromatium vinosum cytochrome c' studied using native mass spectrometry and fluorescence resonance energy transfer.
Cytochrome c' from Allochromatium vinosum is an attractive model protein to study ligand-induced conformational changes. This homodimeric protein dissociates into monomers upon binding of NO, CO or CN(-) to the iron of its covalently attached heme group. While ligand binding to the heme has been well characterized using a variety of spectroscopic techniques, direct monitoring of the subsequent monomerization has not been reported previously. Here we have explored two biophysical techniques to simultaneously monitor ligand binding and monomerization. Native mass spectrometry allowed the detection of the dimeric and monomeric forms of cytochrome c' and even showed the presence of a CO-bound monomer. The kinetics of the ligand-induced monomerization were found to be significantly enhanced in the gas phase compared with the kinetics in solution, however. Ligand binding to the heme and the dissociation of the dimer in solution were also studied using energy transfer from a fluorescent probe to both heme groups of the protein. Comparison of ligand binding kinetics as observed with UV-vis spectroscopy with changes in fluorescence suggested that binding of one CO molecule per dimer could be sufficient for monomerization.
Evers
Toon H
TH
Laboratory of Macromolecular and Organic Chemistry, Department of Biomedical Engineering, Eindhoven University of Technology, Post Office Box 513, 5600 MB Eindhoven, The Netherlands.
van Dongen
Joost L J
JL
Meijer
E W
EW
Merkx
Maarten
M
D001665:Binding Sites; D002248:Carbon Monoxide; D003486:Cyanides; D045305:Cytochromes c' / Q000737:chemistry*; D019281:Dimerization; D031541:Fluorescence Resonance Energy Transfer; D006418:Heme / Q000378:metabolism; D007700:Kinetics; D008024:Ligands; D013058:Mass Spectrometry; D009569:Nitric Oxide; D011487:Protein Conformation
D003486:Cyanides; D045305:Cytochromes c'; D008024:Ligands; D009569:Nitric Oxide; D006418:Heme; D002248:Carbon Monoxide