The 40s Omega-loop plays a critical role in the stability and the alkaline conformational transition of cytochrome c.
The structural and redox properties of a non-covalent complex reconstituted upon mixing two non-contiguous fragments of horse cytochrome c, the residues 1-38 heme-containing N-fragment with the residues 57-104 C-fragment, have been investigated. With respect to native cyt c, the complex lacks a segment of 18 residues, corresponding, in the native protein, to an omega (Omega)-loop region. The fragment complex shows compact structure, native-like alpha-helix content but a less rigid atomic packing and reduced stability with respect to the native protein. Structural heterogeneity is observed at pH 7.0, involving formation of an axially misligated low-spin species and consequent partial displacement of Met80 from the sixth coordination position of the heme-iron. Spectroscopic data suggest that a lysine (located in the Met80-containing loop, namely Lys72, Lys73, or Lys79) replaces the methionine residue. The residues 1-38/57-104 fragment complex shows an unusual biphasic alkaline titration characterized by a low (p K(a1)=6.72) and a high p K(a)-associated state transition (p K(a2)=8.56); this behavior differs from that of native cyt c, which shows a monophasic alkaline transition (p K(a)=8.9). The data indicate that the 40s Omega-loop plays an important role in the stability of cyt c and in ensuring a correct alkaline conformational transition of the protein.
Caroppi
Paola
P
Dipartimento di Medicina Sperimentale e Scienze Biochimiche, Università di Roma Tor Vergata, via Montpellier 1, 00133, Rome, Italy.
Sinibaldi
Federica
F
Santoni
Elisa
E
Howes
Barry D
BD
Fiorucci
Laura
L
Ferri
Tommaso
T
Ascoli
Franca
F
Smulevich
Giulietta
G
Santucci
Roberto
R
D002384:Catalysis; D002942:Circular Dichroism; D003574:Cytochrome c Group / Q000737:chemistry* / Q000378:metabolism; D004563:Electrochemistry; D004795:Enzyme Stability; D006418:Heme / Q000737:chemistry* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007501:Iron / Q000737:chemistry* / Q000378:metabolism; D008239:Lysine / Q000737:chemistry; D008715:Methionine / Q000737:chemistry; D010084:Oxidation-Reduction; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D013059:Spectrum Analysis, Raman
D003574:Cytochrome c Group; D010446:Peptide Fragments; D006418:Heme; D008715:Methionine; D007501:Iron; D008239:Lysine