It played an important role in the detection of mitochondrial apoptosis that the interaction of Cytochrome(Cyt c) with NO, thus the research is still a hotspot issue for the chemists and biologists. In this paper, Ultraviolet (UV-Vis) absorbance spectra, UV-Vis time course spectra, circular dichroism (CD), synchronous fluorescence spectra and electron paramagnetic resonance (EPR) technology were used to research the coordination reaction mechanism of different valence states Cyt c with proliNO/NO and its spatial conformation changes. The results showed that Cyt c could directly react with NO without other reagents. And its secondary structure would change during the experiment. It has been established that the electronic configurations of iron ions in porphyrin complexes are controlled in the nature by a number of axial ligands, peripheral substituents attached to the porphyrin macrocycle, deformation of the porphyrin ring and solvent effects. During the experiment, proliNO/NO was added to the sample of Cyt c, and NO gas would generate, then entered into the solution, finally the distal methionine heme ligand was displaced, Cyt c could bind NO, that is, the Fe—S broke down, and the Fe-N formed. At the same time, generating a new cytochrome c-NO (Cyt c-NO) complex. Cyt c-NO binary complexes were instability and would dissociate, and dissociation process of Cyt c-NO binary complexes was belonging to a first-order reaction with the dissociation rate of (0.071 1±0.039 6) s-1. The secondary structure of Cyt c was affected by proliNO/NO concentration. When the concentrate of proliNO/NO was below to 8.6×10-4 mol·L-1, the peak changes of 222 nm and 208 nm was very weak, and the α-helix increased from 33.1% to 44.1%. And it continued to increase, the secondary structure of Cyt c took place a great change. The tiny changes illustrated that a new compound generated, but excessive proliNO/NO can break the structure of Cyt c. Taken together, we have demonstrated that an understanding of the interaction mechanism of NO with cytc, and the reaction mechanism of NO with ferri- and ferro-cytochrome c have important implications for the inhibition of mitochondrial oxygen consumption by NO and the mitochondrial metabolism of NO.