The final interprotein electron transfer (ET) in the mammalian respiratory chain, from cytochrome
c
(Cyt
c
) to cytochrome
c
oxidase (C
c
O) is investigated by
1
H-
15
N heteronuclear single quantum coherence spectral analysis. The chemical shift perturbation in isotope-labeled Cyt
c
induced by addition of unlabeled C
c
O indicates that the hydrophobic heme periphery and adjacent hydrophobic amino acid residues of Cyt
c
dominantly contribute to the complex formation, whereas charged residues near the hydrophobic core refine the orientation of Cyt
c
to provide well controlled ET. Upon oxidation of Cyt
c
, the specific line broadening of N-H signals disappeared and high field
1
H chemical shifts of the N-terminal helix were observed, suggesting that the interactions of the N-terminal helix with C
c
O are reduced by steric constraint in oxidized Cyt
c
, while the chemical shift perturbations in the C-terminal helix indicate notable interactions of oxidized Cyt
c
with C
c
O. These results suggest that the overall affinity of oxidized Cyt
c
for C
c
O is significantly, but not very much weaker than that of reduced Cyt
c
. Thus, electron transfer is gated by dissociation of oxidized Cyt
c
from C
c
O, the rate of which is controlled by the affinity of oxidized Cyt
c
to C
c
O for providing an appropriate electron transfer rate for the most effective energy coupling. The conformational changes in Lys13 upon C
c
O binding to oxidized Cyt
c
, shown by
1
H- and
1
H,
15
N-chemical shifts, are also expected to gate intraprotein ET by a polarity control of heme
c
environment.