Structural changes of heme-heme oxygenase-1 complex following carbon monoxide (CO) dissociation were studied by time-resolved resonance Raman spectroscopy. We observed temporal changes for resonance Raman bands of the Fe–His stretch and the heme propionate bends in the subnanosecond and microsecond time regimes. These changes suggest structural rearrangements in the Fe–His linkage and the salt bridges of the heme propionates following CO dissociation. The present data supports the model proposed by an X-ray crystallographic study. The Fe–His stretching mode exhibited an upshift until 30 µs after dissociation as the delay time increased. This is the first example of a CO-dissociation-induced strengthening of the Fe-His linkage in hemeproteins. Copyright © 2010 John Wiley & Sons, Ltd.