Interaction of Yeast Iso-1-cytochrome c with Cytochrome c Peroxidase Investigated by [¹⁵N,¹H] Heteronuclear NMR Spectroscopy

Abstract

The interaction of yeast iso-1-cytochrome c with its physiological redox partner cytochrome c peroxidase has been investigated using heteronuclear NMR techniques. Chemical shift perturbations for both 15N and 1H nuclei arising from the interaction of isotopically enriched 15N cytochrome c with cytochrome c peroxidase have been observed. For the diamagnetic, ferrous cytochrome c, 34 amides are affected by binding, corresponding to residues at the front face of the protein and in agreement with the interface observed in the 1:1 crystal structure of the complex. In contrast, for the paramagnetic, ferric protein, 56 amides are affected, corresponding to residues both at the front and toward the rear of the protein. In addition, the chemical shift perturbations were larger for the ferric protein. Using experimentally observed pseudocontact shifts the magnetic susceptibility tensor of yeast iso-1-cytochrome c in both the free and bound forms has been calculated with HN nuclei as inputs. In contrast to an earlier study, the results indicate that there is no change in the geometry of the magnetic axes for cytochrome c upon binding to cytochrome c peroxidase. This leads us to conclude that the additional effects observed for the ferric protein arise either from a difference in binding mode or from the more flexible overall structure causing a transmittance effect upon binding.

NMR basis for interprotein electron transfer gating between cytochrome c and cytochrome c oxidase

“…In addition, the previous NMR study reported a distinct difference between reduced and oxidized Cyt c in terms of the size of the interaction site for CcP (25), but the size of the interaction site for CcO is essentially independent of the redox state (Fig. 3).…”

“…1B), amino acid residues located near the heme moiety such as Glu16, Thr28, Lys72, and Phe82 in the CcP interaction site (21,25) are not involved in interactions with CcO. The contribution of the N-and C-terminal helix regions to regulation of the binding orientation of Cyt c is more prominent in CcO than that in the interaction with CcP (Fig.…”

“…plexes Involving Cytochrome c. The Cyt c-cytochrome c peroxidase (CcP) complex has been the most extensively analyzed ET complex at present (20,21,24,25). The similarity in the complex formation mechanism in the CcO∕Cyt c and CcP∕Cyt c systems has been proposed, because the interaction sites for Cyt c in both CcP and CcO surfaces are surrounded by negatively charged amino acid residues.…”

A further investigation of the cytochrome b 5–cytochrome c complex

“…The second interaction region appears instead to be more susceptible to variations in primary sequence. In our case, the most likely additional binding site comprises helix a 5 (55)(56)(57)(58)(59)(60)(61) and the turn up to residue 64.…”

“…For cytochrome c there is no available literature relative to the effect of the interaction with cytochrome b 5 on the chemical shifts of its backbone nuclei. A chemical shift mapping study is instead available on the interaction between oxidized and reduced 15 N-enriched yeast iso-1 cytochrome c and unlabelled CcP in the so-called resting state [60]. The largest chemical shift variations were observed for residues 12-16 and the region around Phe82.…”