Solution Structure of Horse Heart Ferrocytochrome c Determined by High-Resolution NMR and Restrained Simulated Annealing

Abstract

A model for the solution structure of horse heart ferrocytochrome 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 1940 distance constraints based on the observed magnitude of nuclear Overhauser effects and 85 torsional angle restraints based on the magnitude of determined J-coupling constants. The all-residue root mean square deviation about the average structure is 0.47 +/- 0.09 A for the backbone N, C alpha, and C' atoms and 0.91 +/- 0.07 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. However, a detailed comparison between the model for the solution structure and the available model for the crystal structure of tuna ferrocytochrome c indicates significant differences in a number of secondary and tertiary structural features. For example, two of the three main helices display 3(10) to alpha-helical transitions 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 previously reported models. The geometry of heme ligation is well-defined and completely consistent with the crystal structures of homologous cytochromes c as are the locations of four of six structural water molecules. Though the total solvent-accessible surface area of the protoporphyrin ring is similar to that seen in crystal studies of tuna ferrocytochrome c, the distribution is somewhat different. This is mainly due to a difference in packing of residues Phe-82 and Ile-81 such that Ile-81 crosses the edge of the heme in the solution structure. These and other observations help to explain a range of physical and biological data spanning the redox properties, folding, molecular recognition, and stability of the protein.

Solution Structure of Oxidized Horse Heart Cytochrome c,

“…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). 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.…”

“…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). We felt that some of the differences could be ascribed to the poor resolution of the solution structure of the oxidized species due to the lack of few strategic experimental constraints.…”

“…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. The 5-14, 49-55, 60-68, 91-103 R-helices and a 70-74 3 10 -helix are observed in the reduced protein, which compare to the R-helices 2-7, 9-14, 49-54, 60-69, 89-102 and the 3 10 helix 70-74 of the present structure.…”

Conformations of biopolymers in the gas phase: a new mass spectrometric method

“…This unfolding is substantial for the charge states reported here since the calculated cross section of the x-ray structure [17] and the solution NMR structure [18] is about 1350 Å 2 [12]. However, the results of Shelimov et al indicate the lowest charge states they investigated (3ϩ to 5ϩ) are substantially more compact than the native structure with cross sections of ϳ1200 Å 2 .…”

Determinants of protein hydrogen exchange studied in equine cytochrome c

“…Structural comparisons made here utilized the crystal structure of oxidized horse cyt c refined to 1.9 8, (Bushnell et al, 1990) (PDB#lHRC) and the solution NMR structures (Qi et al, 1994a(Qi et al, , 1996 (PDB#lFRC and A.J. Wand, pers.…”