Haem proteins conformation change

The reduction of ferricytochrome c by at neutral pH appears to be a three-step process. In the first step (A =4.5x 10 lmol- s ) a transient complex is formed between the cytochrome and the hydrated electron, in the second (k= 5 X 10 s ) the haem iron is reduced, and in the third (/ = 1.3 x 10 s ) the protein conformation changes from that appropriate for Fe to that appropriate for Fe. The authors favour a specific pathway for the movement of the electron from the surface of the molecule to the haem iron (step 1). No intermediate complexes were observed in the reduction of ferricytochrome c by the superoxide radical ion. At 20 °C the rate constant for the reaction at pH 4.7—6.7 is 1.4 x 10 1 mol s and as the pH increases above 6.7 the rate constant steadily decreases (eventually reaching zero, indicating that the neutral and high-pH forms if ferricytochrome c are un-reactive). The activation enthalpy is 18 kJ mol and it seems that little protein rearrangement is required for the formation of the activated complex. The kinetics have been reported for the reduction by Cr + of 2-hydroxy-5-nitrobenzyltryptophyl cytochrome c and of iV-formyltryptophyl cytochrome c. ... 

The rate of enzyme turnover is usually determined, not simply by the duration of the substrate-product transformation at the enzyme active center, but by the rate of protein globule relaxation. There is much experimental evidence for this statement. For example, a kinetic and structural study of cytochrome C modified by the ruthenium label specifically at the imidazole moiety of histidine 33, demonstrated that the rate of intramolecular electron transfer, Ru(2) - Haem(3), k 55 s" [56], is on the same time scale as the rate of conformational changes occurring within the cytochrome C molecule [57, 58]. Together with other experimental results (see, for references [38, 39]), this is an obvious indication of the protein conformational changes as the rate-limiting step in protein functioning. 

Cyt c is associated with the outer surface of the inner mitochondrial membrane. Phospholipids induce conformational changes in the protein and, in certain instances, the haem can convert to the high spin (S = 5/2) form, indicative of a weakening of the ligand field caused by displacement of the sixth ligand (Met-80). This has been associated with the detection of lipid radicals by direct EPR (at 11 K).65 Indeed, peroxidase-type activity is also evident in the reaction of cyt c with lipid hydroperoxides, as studied by spin trapping in conjunction with HPLC and MS.66... 

Diphasic kinetic behaviour in the reduction of cyt c(iii) by glutathione, which has the potential to recognize different protein forms, suggests - the existence of two cyt c(iii) conformers which may be interconverted by protonation (pl ra 7) followed by a slow conformational change. Transient glutathione-cyt c(iii) complexes were detected at the 339 nm isosbestic and the retention of the 695 nm absorption indicates that the site of complexation is not close to the haem iron. Decay of the transient at 339 nm is consistent with slow disulphide formation. 

---