Protein dynamics of isolated chains of recombinant human adult hemoglobin (rHb) following ligand photolysis were studied by time-resolved resonance Raman spectroscopy. In the time-resolved spectra, we observed frequency shifts of the iron-histidine stretching [nu(Fe-His)] and gamma(2) bands and an intensity change of the nu(8) band for both the isolated alpha- and beta-chains, showing that a primary metastable form was present in the initial tens of nanoseconds following the photolysis. Similar spectral changes were reported for human adult hemoglobin and rHb. Common spectral changes between isolated chains and hemoglobin indicated that structural changes reflected by the spectral changes were characteristic of the hemoglobin subunits. The heme modes suggested that the primary metastable form had a more disordered orientation of propionates and a less strained environment than the deoxy form. The spectral changes of the isolated a-chain were faster than those of the beta-chain. In spite of the fact that the isolated beta-chain formed a tetramer in a similar fashion to rHb, the spectral changes were much slower than those of rHb. The present study shows that intersubunit interactions affected the rates of the structural changes of the heme pocket. Characteristics of the tertiary structure dynamics of hemoglobin are discussed.