  In haemoglobin (consisting of four globular myoglobin-like subunits), the
change from the low-spin (LS) hexacoordinated haem to the high spin (HS)
pentacoordinated domed form upon ligand detachment and the reverse process upon
ligand binding, represent the transition states that ultimately drive the
respiratory function. Visible-ultraviolet light has long been used to mimic the
ligand release from the haem by photodissociation, while its recombination was
monitored using time-resolved infrared to ultraviolet spectroscopic tools.
However, these are neither element- nor spin-sensitive. Here we investigate the
transition state in the case of Myoglobin-NO (MbNO) using femtosecond Fe Kalpha
and Kbeta non-resonant X-ray emission spectroscopy (XES) at an X-ray
free-electron laser upon photolysis of the Fe-NO bond. We find that the
photoinduced change from the LS (S = 1/2) MbNO to the HS (S = 2)
deoxy-myoglobin (deoxyMb) haem occurs in ca. 800 fs, and that it proceeds via
an intermediate (S = 1) spin state. The XES observables also show that upon NO
recombination to deoxyMb, the return to the planar MbNO ground state is an
electronic relaxation from HS to LS taking place in ca. 30 ps. Thus, the entire
ligand dissociation-recombination cycle in MbNO is a spin cross-over followed
by a reverse spin cross-over process.
