Cytochrome c oxidase (CcO), the terminal enzyme in the electron transfer chain, translocates protons across the inner mitochondrial membrane by harnessing the free energy generated by the reduction of oxygen to water. Several mechanisms for this redox coupling have been proposed, but they lack confirmation, in part from uncertainties in the reported crystal structures due to radiation damage effects caused by the intense synchrotron radiation. Here, we report the room temperature damage-free structure of the carbon monoxide (CO) bound derivative of bovine CcO (CO-bCcO) obtained by serial femtosecond X-ray crystallography (SFX) with an X-ray free electron laser and compare it to a CO-bCcO structure obtained with a synchrotron light source. In the SFX structure, resolved at 2.3 Å, the CO is coordinated to the heme a3 iron atom and is bent to 134⁰ from the heme plane, whereas in the structure of CO-bCcO obtained by synchrotron radiation, at a resolution of 1.95 Å, the Fe-CO bond is cleaved and the CO has moved to a position near CuB. Associated with CO dissociation from heme a3, the distance between the heme a3 iron atom and CuB is reduced from 5.27 to 4.91 Å, primarily owing to a change in position of CuB. An allosteric transition, involving a large movement of a section of the Helix-X polypeptide that lies between the two hemes, is triggered by the change in ligation state. This transition plays a critical role in postulated mechanisms of proton translocation in CcO.