pH-dependent equilibria of yeast Met80Ala-iso-1-cytochrome c probed by NMR spectroscopy: a comparison with the wild-type protein.
BACKGROUND
Cytochrome c has five distinct pH-dependent conformational states, including two alkaline forms of unknown structure. It is believed that in both of the alkaline forms a Lys residue is ligated to the heme, but the identity of the Lys residue is different. Exchange between these forms would require extensive structural rearrangement. Mutation of the heme axial ligand (Met80) to Ala in Saccharomyces cerevisiae iso-1-cytochrome c yields a protein (Ala80cyt c) capable of binding exogenous ligands such as dioxygen and cyanide. We have analyzed the 1H NMR spectra of this mutant at various pH values in the hope of gaining insight into the structure of the acidic and alkaline forms of native cytochrome c.


RESULTS
The pH dependence of the 1H NMR spectrum of ferriAla80cyt c is consistent with the high-spin/low-spin transition (pKa = 6.5) observed by absorption spectroscopy. The T1 values for the low-spin form are consistent with OH ligation, as inferred previously from absorption and electron paramagnetic resonance spectroscopic results. The pH-dependent equilibria of ferriAla80cyt c differ from those of the wild-type protein. Both Ala80 and wild-type ferricyt c appear to have the same iron coordination at low pH (approximately equal to 2), while only one alkaline form of Ala80cyt c (versus two for WTcyt c) was detected.


CONCLUSIONS
The differences between the pH dependence of the 1H NMR spectra of Ala80cyt c and those of the wild-type protein demonstrate that the heme axial ligands influence the relative energies of the conformational states of cytochrome c. The results are consistent with the notion that a large rearrangement is required to switch between the two alkaline forms.
Banci
L
L
Department of Chemistry, University of Florence, Italy.
Bertini
I
I
Bren
K L
KL
Gray
H B
HB
Turano
P
P
D003574:Cytochrome c Group / Q000737:chemistry / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy; D006863:Hydrogen-Ion Concentration; D007501:Iron / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy; D008715:Methionine / Q000737:chemistry / Q000378:metabolism*; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae / Q000201:enzymology*
D003574:Cytochrome c Group; D008715:Methionine; D007501:Iron