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

In the accompanying paper [Ferraiolo, B. L., Onady, G. M., & Mieyal, J. J. (1984) Biochemistry (preceding paper in this issue)] we reported different aniline hydroxylase activities for 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 reduced nicotinamide adenine dinucleotide phosphate (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 hemoglobin (Hb) species. The paramagnetic contribution of the ferric heme iron 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 two 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.(ABSTRACT TRUNCATED AT 250 WORDS)