Methionine ligand lability of type I cytochromes c: detection of ligand loss using protein film voltammetry.
Protein film voltammetry (PFV) is used to interrogate the behavior of a variety of bacterial and mitochondrial His/Met-ligated cytochromes c. While analogous studies upon alkanethiol-modified gold electrodes reveal the anticipated Fe(II/III) couple only, PFV using pyrolytic graphite edge (PGE) electrodes demonstrates the presence of a lower-potential form of each of the cyts c studied, with a potential of approximately -100 mV (vs hydrogen). The generation of the novel, lower-potential state is shown to arise specifically from the interaction with the PGE electrode. Simultaneously, the typical Fe(II/III) couple can be observed. PFV of a series of wild-type cytochromes and mutants in the Met-donating loop show that the lower-potential state is highly similar between proteins from Pseudomonas aeruginosa (PA), Hydrogenobacter thermophilus (HT), and horse heart. The generation of the lower-potential form correlates inversely with the stability of the Met-Fe interaction for each of the cytochromes. Comparison with chemically unfolded cyts c indicates that the lower-potential forms detected here are unique, and this distinct state is ascribed to the loss of the Met ligand. Thus, PGE is demonstrated to be a non-innocent electrode surface in PFV studies of His/Met-ligated cytochromes c.
Ye
Tao
T
Departments of Chemistry, Boston University, 590 Commonwealth Avenue, Boston, Massachusetts 02215, USA.
Kaur
Ravinder
R
Senguen
F Timur
FT
Michel
Lea V
LV
Bren
Kara L
KL
Elliott
Sean J
SJ
D000818:Animals; D045304:Cytochromes c / Q000737:chemistry*; D004563:Electrochemistry / Q000379:methods*; D004566:Electrodes; D006108:Graphite / Q000737:chemistry; D008024:Ligands; D008715:Methionine / Q000737:chemistry*; D011550:Pseudomonas aeruginosa
D008024:Ligands; D006108:Graphite; D045304:Cytochromes c; D008715:Methionine