PH-DEPENDENCE OF STRUCTURAL AND FUNCTIONAL-PROPERTIES OF OXIDIZED CYTOCHROME-C'' FROM METHYLOPHILUS-METHYLOTROPHUS

Cytochrome c '' from Methylophilus methylotrophus is an unusual monohe me protein that undergoes a major redox-linked change in the heme arra ngement: one of the two axial histidines bound to the iron in the oxid ized form is detached upon reduction and a proton is taken up. The kin etics of reduction by sodium dithionite and the spectroscopic properti es of the oxidized cytochrome c '' have been investigated over the pH range between 1.4 and 10.0, The rate of reduction displays proton-link ed transitions of pK(a) congruent to 5.5 and 2.4, and a spectroscopic transition with a pK(a) congruent to 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 sug gest that the transition at lower pH brings about a drastic change in the heme coordination geometry, Circular dichroism spectra indicate th at over the same proton-linked transition, the pro tein undergoes a ma rked decrease (similar to 60%) of the alpha-helical content toward a r andom coil arrangement, which is recovered upon increasing the ionic s trength, 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 r emarkable capacity of this protein to recover its native structure aft er exposure to extremely low pH values makes it a promising model for studying folding processes and stability in heme proteins.