Monooxygenase activity of human hemoglobin: NMR demonstration of different modes of substrate binding corresponding to different activities of hemoglobin derivatives

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Abstract
Different aniline hydroxylase activities are exhibited by ferrihemoglobin, its isolated subunits, and the converse pair of valency hybrids .alpha.3+2(.beta.2+-CO)2 and (.alpha.2+-CO)2.beta.3+2 in a reconstituted system containing NADPH and cytochrome P-450 reductase. To investigate the molecular basis for the different activities, 1H NMR T1 relaxation studies of aniline were performed in the absence and presence of each of the Hb species. The paramagnetic contribution of the ferric heme Fe atoms of each Hb derivative to the enhanced relaxation of the proton nuclei of aniline was determined relative to control experiments in which the hemoproteins had been converted fully to the corresponding (carbonmonoxy)ferrous forms, which are diamagnetic. According to the known distance dependence of the paramagnetic effect and the relative changes in T1 for the upfield and downfield signals in the spectrum of aniline, it was ascertained that aniline binds in the same manner to the .beta.-ferric hybrid and to ferrihemoglobin. These 2 forms displayed equivalent hydroxylase activities that were the highest among the Hb derivatives for the same aniline concentration. The T1 changes observed with the .alpha.-ferric hybrid suggest a different orientation for aniline in that complex. The T1 data for the isolated subunits .alpha.3+ and .beta.3+4 would indicate that overall binding of aniline includes a component of direct aniline-heme ligation in each case. For .beta.3+4 this result is consistent with the marked substrate inhibition observed for the hydroxylase activity. The specific mode of substrate association near the heme may determine the efficiency of monooxygenase catalysis.