pH dependence of structural and functional properties of oxidized cytochrome c" from Methylophilus methylotrophus.
Cytochrome c" from Methylophilus methylotrophus is an unusual monoheme protein that undergoes a major redox-linked change in the heme arrangement: one of the two axial histidines bound to the iron in the oxidized form is detached upon reduction and a proton is taken up. The kinetics of reduction by sodium dithionite and the spectroscopic properties of the oxidized cytochrome c" have been investigated over the pH range between 1.4 and 10.0. The rate of reduction displays proton-linked transitions of pKa congruent with 5.5 and 2.4, and a spectroscopic transition with a pKa congruent with 2.4 is also observed. The protein displays a complete reversibility after exposure to low pH, and both electronic absorption and resonance Raman spectroscopic properties suggest that the transition at lower pH brings about a drastic change in the heme coordination geometry. Circular dichroism spectra indicate that over the same proton-linked transition, the protein undergoes a marked decrease (approximately 60%) of the alpha-helical content toward a random coil arrangement, which is recovered upon increasing the ionic strength. The structural change at low pH is linked to a concerted two-proton transition, suggesting the detachment and protonation of axial histidine(s). Such kinetic and spectroscopic features along with the remarkable capacity of this protein to recover its native structure after exposure to extremely low pH values makes it a promising model for studying folding processes and stability in heme proteins.
Coletta
M
M
Department of Experimental Medicine and Biochemical Sciences, University of Roma Tor Vergata, Via di Tor Vergata 135, I-00133 Roma, Italy.
Costa
H
H
De Sanctis
G
G
Neri
F
F
Smulevich
G
G
Turner
D L
DL
Santos
H
H
D002942:Circular Dichroism; D003574:Cytochrome c Group / Q000737:chemistry* / Q000378:metabolism*; D006088:Gram-Negative Aerobic Bacteria / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010084:Oxidation-Reduction; D011487:Protein Conformation; D013053:Spectrophotometry; D013059:Spectrum Analysis, Raman
D003574:Cytochrome c Group; C076615:cytochrome c''