A possible structure for the higher oxidation state of metmyoglobin.

In a previous paper (George & Irvine, 1954a) we concluded that there are two kinds of structure for the higher oxidation state of metmyoglobin (MetMb) which could explain the experimental observations. One is a radical structure formed by hydrogen atom removal from a methine carbon atom, a pyrrolic carbon atom or some other atom within the conjugated network of porphyrin ring and haemoprotein linkage; the other is a derivative of quadrivalent iron of the ferryl ion type, FeO2+. Further experiments have been undertaken to enable a choice to be made between these two structures, and the results ofthese are described and discussed below. The experiments fall into two sections. In the first, the hydrogen-ion changes accompanying the formation ofthe higher oxidation state in unbuffered solutions were measured, while in the second, the equilibrium constant for the reaction between MetMb and K2IrCl was determined under varying conditions ofpH, temperature and ionic strength. The results of both classes of experiments are most simply interpreted on the basis of the ferryl ion structure. A brief account of the pH measurements has already been published (George & Irvine, 1954 b). MATERIALS AND METHODS Pota88ium chloroiridate. This was the same as that used previously (George & Irvine, 1954a). Spectrophotometric and potentiometric titrations with K4Fe(CN)6 showed the sample to be 99% pure. The titrations were carried out in acid solution using glass-distilled HCl, and the solutions were made up with water distilled from dil. KMnO4. In this way reduction of the E,IrC0 by trace reducing matter was prevented. In the experiments using unbuffered solutions, the EKIrCl was made up in C02-free water as described below, and used immediately to minimize any effect resulting from its hydrolysis. In the equilibrium experiments K2IrCl was made up in dilute glass-distilled HC1 (10-4M) to prevent hydrolysis, although, as was shown previously (George & Irvine, 1954a) if hydrolysis occurs to any extent, its effect is almost negligible. The strength and volumes of the buffer solutions used were such that their pH values were not affected by the acid in the K2IrCl4 solution. Buffer solution8. In the experiments with K2IrCls, phosphate buffers (NaOH +NaH,P04) were used, the ionic strengths being adjusted to the value required by the addition ofAR NaCl. The pH ofeach solution was measured using a Cambridge pH meter calibrated with 0-05m potassium hydrogen phthalate buffer. Stock solutions of approximately 0-IN were prepared by dilution, standardized against KMnO4, and then …