The Nature of the Thermal Equilibrium Affecting the Iron Coordination of Ferric Cytochrome c

Abstract

In cytochrome c, ligation of the heme iron by the methionine-80 sulfur plays a major role in determining the structure and the thermodynamic stability of the protein. In the ferric state, this bond is reversibly broken by moderately acid or alkaline pH's (pK's 2.5 and 9.4, respectively) and by exogenous ligands. NMR studies of horse ferricytochrome c in which the Met-65 and Met-80 methyl groups were chemically enriched with 13C demonstrate that, at 59 degrees C, a temperature at which the protein is still folded, the sulfur-iron bond is already partially broken. This structural change corresponds to the reversible disappearance upon moderate heating of the 695 nm band, characteristic of the sulfur-iron coordination of this protein. The thermal effect results from a shift in the alkaline pK from 9.4 at 25 degrees C to 8.2 at 59 degrees C. The exchange rate from iron-bound to free methionine-80 at 59 degrees C is 1.8 s-1, as measured by saturation transfer experiments. The free and bound methionine-80 epsilon-methyl groups in the 1H spectrum are assigned as (1.87, 2.25) and -21.43, respectively; in the 13C spectrum they are assigned as 15.6 and 12.8, respectively (all these values are in ppm from 3-(trimethylsilyl)propionic-2,2,3,3-d4 acid, sodium salt).

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

“…The number of assigned NOEs is 2228 for the minor B species and 2267 for the major A species, including the nonsplit cross peaks assigned for both species. Consistent with what is observed for the splitting of the signals, sizable differences in NOE couplings are observed for residues 15,16,17,18,19,29,30,31,35,64,81,82,85,90,91, 95 and 98.…”

“…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 . Furthermore, ligands such as NH 3 [20] and imidazole [21,22] have been shown to substitute the methionine ligand.…”

Fast Coordination Changes in Cytochrome c Do Not Necessarily Imply Folding

“…In native cyt-c, the heme iron is six-coordinate because the polypeptide chain provides two axial ligands, namely a His (His-18, the proximal His; numbering is for the sequence of horse heart cyt-c) and a Met (Met-80) (5,6). The distal Fe-Met bond contributes to a significant extent to the overall thermodynamic stability of native cyt-c (7,8) (in the unfolding process this bond is weakened), and the unfolded cyt-c is a mixture of the fivecoordinate and several six-coordinate states, the sixth ligand being provided either by Met-80 or by other His or Met residues of the polypeptide chain (a phenomenon called miscoordination) (3,4,9,10)). Because of the weakening of the Fe-Met bond, external heme ligands, such as CO, induce unfolding at lower denaturant concentrations by competing with Met-80 for the sixth coordination position.…”

The Conformational Flexibility of Oxidized Cytochrome c Studied through Its Interaction with NH3 and at High Temperatures

“…All these species contain low-spin iron(III). Recently, Taler et al have further characterized the high-temperature species of horse cytochrome c and identified the 1 H-NMR resonances of the ε-CH 3 of Met80 in the high-temperature species at typical diamagnetic shift values [14] .…”