NMR study of the alkaline isomerization of ferricytochrome c

Abstract

The pH-induced isomerization of horse heart cytochrome c has been studied by tH NMR. We find that the transition occurring in D20 with a pKa measured as 9.5+0.1 is from the native species to a mixture of two basic forms which have very similar NMR spectra. The heme methyl peaks of these two forms have been assigned by 2D exchange NMR. The forward rate constant (native to alkaline cytochrome c) has a value of 4.0+0.6 s -~ at 27°C and is independent of pH; the reverse rate constant is pH-dependent. The activation parameters are zlH~ = 12.8 +0.8 keal.moP, z/S~ = -12.9 __+ 2.0 e.u. for the forward reaction and ,Jilt = 6.0_+0.3 kcal.mo1-1, AS~= -35.1 + 1.3 e.u. for the reverse reaction (pH* =9.28).ztH ° and z/S ° for the isomerization are 6.7 + 0.6 kcal.mol -~ and 21.9 + 1.0 e.u., respectively.

Nitration of Solvent-exposed Tyrosine 74 on Cytochrome c Triggers Heme Iron-Methionine 80 Bond Disruption

“…As already reported, spectra recorded at different pH values evidenced the transition from the native low spin form (III) at neutral pH to the alkaline low spin form (IV) (Fig. 4B) (13,18,19,24). Several resonances change at high pH (such as those from methyl groups 3, 5, and 8) revealing an overall perturbation of the heme environment.…”

“…As an alternative to these spectroscopic methods, changes in heme electronic structure in cytochrome c 3ϩ can be monitored by looking at the paramagnetically shifted resonances in 1 H NMR spectra (33)(34)(35), as both species are low spin and give sharp, sensitive signals, with different chemical shifts under a slow exchange regime (13,19). NMR not only allows the characterization of the different conformations but also assists in the determination of the pK a for the observed transition, which can be followed at the residue level (19,35). Moreover, cytochrome c 3ϩ -cardiolipin interactions have been evaluated preliminarily by 1 H-NMR (36).…”

NMR investigation of ferricytochrome c unfolding: Detection of an equilibrium unfolding intermediate and residual structure in the denatured state

“…Changes in heme substituent shifts for cyt c, therefore, are attributed to replacement of Met-80 with non-native ligands, whereas the His-18 maintains its native orientation. The pattern of heme methyl shifts for species L bears a striking resemblance to that observed for the alkaline form of cyt c, known to have two conformers in which the axial Met ligand is replaced with Lys side chains (Table 1) (33,38). It also bears a strong similarity to the cyanide adducts of Met-80-Ala cyt c (34) and MP-8 (37).…”

“…It also bears a strong similarity to the cyanide adducts of Met-80-Ala cyt c (34) and MP-8 (37). Species L, alkaline cyt c, and these cyt c derivatives all have heme methyl shifts ranging from 25 to 10 ppm with an order of 8-CH 3 Ͼ 5-CH 3 Ͼ 1-CH 3 Ͼ 3-CH 3 , as well as an upfield-shifted heme ␦-meso resonance (Table 1) (32)(33)(34)(35)(36)(37). Its similarities with the NMR properties of these characterized paramagnetic proteins indicate that the cyt c unfolding intermediate in urea is ligated by the native His-18 and a second ligand with cylindrical symmetry.…”

Monitoring the conformational flexibility of cytochrome c at low ionic strength by 1H‐NMR spectroscopy

“…When the oxidized form is dissolved in water, it gives rise to an alkaline form [3Ϫ17] in which the axial ligand methionine residue is detached from the coordination to iron(III). The amount of the alkaline form is pH dependent with a pK a of 9.5 [16]. At temperatures higher than 320 K and neutral pH, other species are detected, again with the axial methionine residue detached from coordination [18,19], which experience a somewhat lower pK a .…”