The nature of ferrous haem protein complexes prepared by photolysis

Abstract

Ultra-low-temperature magnetic circular dichroism (MCD) spectroscopy has been used to investigate the ground-state magnetic properties of deoxymyoglobin, -haemoglobin and reduced cytochrome c oxidase, all of which contain the high-spin ferrous haem ( S = 2). In addition the MCD properties have been studied of the fully photolysed forms, frozen at 1.5 K, of the carbonmonoxy derivatives of these three haemoproteins. In these cases the use of very low temperatures prevents both the thermal recombination of CO and haem and the relaxation of the haem and its associated protein-bound ligand, imidazole. MCD spectra and magnetisation curves show clearly that the state of the haem produced by photolytic removal of the CO ligand and prior to relaxation of the haem centre is the high-spin ( S = 2) ferrous form. However, the zero-field splitting parameters of the ground states of the deoxy and photolysed-CO forms of Hb are quantitatively different. This attests to the presence of significant conformational changes at the haem which occur after the sixth ligand has been removed. The same is found by comparison of the MCD of deoxy-Hb and photolysed-CO-Hb. This is not unexpected, since various physical techniques have revealed that the ligand-bound and deoxy conformations of these two proteins are different at the haem site. We have shown that MCD spectroscopy can detect this difference sensitively. However, in marked contrast the MCD properties of the high-spin ferrous ( S = 2) cytochrome a 3in the reduced and the photolysed CO form of cytochrome c oxidase are quite indistinguishable. This is attributed to the absence of any significant structural or conformational change at the cytochrome a 3 site when the ligand CO becomes bound (or unbound). This suggests that there is no iron movement in- or out-of-plane or any haem doming in cytochrome a 3 when CO is photolysed off. These observations account for the lower binding affinity of CO for cytochrome c oxidase and the absence of a ‘quickly reacting’ form of this enzyme.