The pH dependence for the interconversion of the acid and base forms of methemerythrin from Themiste dyscritum was investigated by difference spectroscopy. A new technique was designed to be able to study mixtures without knowledge of extinction coefficients or exact protein concentrations. The resultant pKa value of 8.4 proved that T. dyscritum hemerythrin crystals used for previous X-ray crystallographic studies at pH less than or equal to 6.5 were in the acid form. Since this material contains a 5-coordinate iron atom with no evidence of a ligated water molecule, it is more appropriately referred to as methemerythrin than aquomethemerythrin. The presence of an iron-bound hydroxide in the base form of methemerythrin was verified by resonance Raman spectroscopy for both T. dyscritum and Phascolopsis gouldii. At pH greater than 9, the protein from either species exhibited a new feature at 490 cm-1 that shifted to 518 cm-1 in D2O and was assigned to a coupled Fe-OH stretching and O-H bending vibration. Thus, hydroxomethemerythrin is the correct designation for the base form of the protein. The other resonance-enhanced vibration, the Fe-O-Fe symmetric stretch, was observed at 506 cm-1 in hydroxomethemerythrin and at 511 cm-1 in methemerythrin and was unaffected by deuteration. Addition of perchlorate to methemerythrin had no effect on the Raman spectrum, despite its known role in stabilizing the met form relative to the hydroxomet form.
Animals Hemerythrin