Raman spectroscopy of redox proteins


A spectroelectrochemical flow cell was developed for simultaneous study of electrochemical properties and vibrational structures of heme proteins. A Raman microscope with an Ar laser and a recently obtained Kr laser, which allows for 413 nm excitation of the intense Soret bands of the heme group, is crucial for this research. Much effort is put into the development of surface-enhanced resonance Raman spectrosocopy (SERRS)-active silver surfaces, covered with self-assembling mono- and bilayers (alkanethiols and phospholipids) to which the proteins can be bound to avoid denaturation. Electrochemistry is used both for the necessary roughening procedures of the silver surface to get a good SERRS effect, and for cyclic voltametry. Research is currently focussed on the
alkaline transition of cytochrome c, a conformational change of the protein induced by pH changes which is thought to play a role in apoptosis. Within the context of the Chemistry of Complex Molecules (CCM) program, human cytochrome P4502D6, an important enzyme for drug metabolism responsible for the breakdown of about 30% of currently marketed medicins, is studied in collaboration with the Molecular Toxicology group. Detailed information about changes in spin state, coordination state, and oxidation state of the heme Fe, could be obtained for the first time for this protein. Changes of these properties, as well as in other vibrational features (for instance frequencies and intensities of vinyl side chains of the heme) were found upon substrate binding.
Future plans include the development of supported phospholipid bilayers for the study of integral membrane proteins, and a systematic study of silver coated, deposited silica nanospheres, to optimize the conflicting requirements of good electrochemical properties and good surface enhancement.

 
Contact: Gert van der Zwan, e-mail: g.van.der.zwan@few.vu.nl; Cees Gooijer, e-mail: c.gooijer@few.vu.nl
Applied Spectroscopy

SERRS spectrum showing spin marker bands of the Fe-heme moiety in P4502D6 with
and without bound substrate. In the resting state (no substrate bound; purple spectrum)
the Fe is predominantly in the low spin(LS) state, due to binding with water as the sixth
ligand. Substrate binding changes the spin state and redox potential of Fe, making an
electron transfer possible from P450 reductase.