Solution Structure of Horse Heart Ferricytochrome c and Detection of Redox-Related Structural Changes by High-Resolution ¹H NMR^,

Abstract

A model for the solution structure of horse heart ferricytochrome c has been determined by nuclear magnetic resonance spectroscopy combined with hybrid distance geometry-simulated annealing calculations. Forty-four highly refined structures were obtained using a total of 1671 distance constraints based on the observed magnitude of nuclear Overhauser effects and 58 torsion angle restrains based on the magnitude of determined J-coupling constants. The model incorporates six long-lived water molecules detected by pseudo-two-dimensional NOESY-TOCSY spectra. The all-residue root mean square deviation about the average structure is 0.33 +/- 0.04 A for the backbone N, C alpha, and C' atoms and 0.83 +/- 0.05 A for all heavy atoms. The overall topology of the model for solution structure is very similar to that seen in previously reported models for crystal structures of homologous c-type cytochromes though there are a number of significant differences in detailed aspects of the structure. Two of the three main helices display localized irregularities in helical hydrogen bonding resulting in bifurcation of main chain hydrogen bond acceptor carbonyls. The N- and C-terminal helices are tightly packed and display several interhelical interactions not seen in reported crystal models. To provide an independent measure of the accuracy of the model for the oxidized protein, the expected pseudocontact shifts induced by the spin 1/2 iron were compared to the observed redox-dependent chemical shift changes. These comparisons confirm the general accuracy of the model for the oxidized protein and its observed differences with the structure of the reduced protein. The structures of the reduced and oxidized states of the protein provide a template to explain a range of physical and biological data spanning the redox properties, folding, molecular recognition, and stability of the cytochrome c molecule. For example, a redox-dependent reorganization of surface residues at the heme edge can be directly related to the redox behavior of the protein and thereby provides a previously undocumented linkage between structural change potentially associated with molecular recognition of redox partners and the fundamental parameters governing electron transfer.

Solution Structure of Oxidized Horse Heart Cytochrome c,

“…The present results, at variance, show a much higher similarity between the solution and crystal structures of hh cyt c (Bushnell et al, 1990) and with the solution structure of reduced hh cyt c (Qi et al, 1994) ( Figure 5). In the absence of the coordinates and of the experimental structural constraints, we may ascribe the differences between the present structure and that previously reported (Qi et al, 1996) as simply due to the better structure refinement of the present one. Indeed, after running PROCHECK (Laskowski et al, 1993) on the solution structure of reduced hh cyt c (Qi et al, 1994) deposited in the Protein Data Bank, very similar secondary structure elements are observed for the two solution structures.…”

“…In particular, we have found that the two forms of S. cereVisiae cytochrome c in solution (Banci et al, 1997a;Baistrocchi et al, 1996) differ only because of the H-bond network involving propionate-7 and for the orientation of a few side chains which have been proposed to be involved in the interaction with biological redox partners (Pelletier & Kraut, 1992). All the elements of secondary structure are maintained, in accordance with the data on the X-ray structures of S. cereVisiae Langen et al, 1992) and tuna (Takano & Dickerson, 1981b;Takano & Dickerson, 1981a) cytochromes c. Recently, the solution structure of oxidized horse heart cytochrome c (hh cyt c, hereafter) has been solved (Qi et al, 1996). It seems substantially different, even for what concerns the secondary structure, from the available solution structure of the reduced form (Qi et al, 1994) and from the X-ray crystal structure of the oxidized protein (Bushnell et al, 1990).…”

“…The similarity is especially evident for the backbone conformation. Within the above frame the recently published data of Qi et al (Qi et al, 1996) appear quite unexpected and suggest a different behavior for the horse heart cytochrome c with respect to the S. cereVisiae protein. The present results, at variance, show a much higher similarity between the solution and crystal structures of hh cyt c (Bushnell et al, 1990) and with the solution structure of reduced hh cyt c (Qi et al, 1994) ( Figure 5).…”

Covalent Adducts Arising from the Decomposition Products of Lipid Hydroperoxides in the Presence of Cytochrome c

“…It is also highly conserved between species and is well characterized structurally by X-ray crystallography and NMR (15)(16)(17)(18). In addition, its location in the inner membrane of the mitochondria, a highly oxidizing environment, makes it a potential target in vivo.…”

Oxidative modification of cytochrome c by singlet oxygen

“…The presence of unmodified b3-b4 and y4-y14 ions confirms the absence of modifications in residues other than the tryptophan. The crystal structure of horse heart Cyt-c (coordinates obtained from 2FRC in the Protein Data Bank [60]) represented as spheres drawn at 90% of the CPK radius with all atoms colored cyan except as noted. His28 and His33 which are modified by 1 O 2 are differentially colored with carbons gray, hydrogens white, and nitrogen blue.…”