Transient Raman study of CO-haemoprotein photolysis: Origin of the quantum yield

Carbon monoxide and haemoproteins (Fe²⁺) form ligand-protein complexes that are photodissociated with high quantum efficiency by visible light^1,2. By photolysing the CO-protein complex with pulsed laser radiation it is possible to generate transient species with properties which reflect both the nature of the photolytic and recombination processes as well as non-equilibrium protein dynamics. Using a single pulse both to photolyse and examine the sample, we previously reported³ the resonance Raman spectrum of a transient species of haemoglobin (Hb) occurring within 10 ns of the photodissociation of carboxyhaemoglobin (HbCO). Transient species generated by photodissociating HbCO have been intensively studied by using transient absorption spectroscopy covering picosecond^4-6, nanosecond ^7-10 and longer time scales^11-15. The overlap of the absorption bands related to various species can hamper the extraction of quantitative information from absorption spectra. Nevertheless, progress has been reported, in particular, the results of nanosecond transient absorption studies^9,10 reported after the experiments described here were well under way. Those results, including re-interpretation of earlier data ⁸, led to the suggestion^9,10 that photolysis of HbCO actually leads to a near-unity quantum yield of a deoxy-Hb species, followed by substantial recombination within 100 ns. We present here definitive evidence from Raman spectra of the 1,350-1,380-cm^-1 region, where the HbCO and deoxy-Hb species exhibit well separated peaks, that this interpretation is indeed correct. We have also measured accurately the HbCO population as a function of delay time after full photolysis, and thereby measured the kinetics of the recombination process. Our findings bear directly on such questions as geminate recombination and the origin of the difference in quantum yield of photodissociation for HbCO and carboxymyoglobin (MbCO). These two systems exhibit very different quantum yields when strongly pumped in the visible: MbCO undergoes 97% photolysis whereas HbCO undergoes only 45-47% (ref. 15).