SULFMYOGLOBIN DERIVED FROM DEUTEROHEMIN RECONSTITUTED PROTEIN .1. STRUCTURE OF THE INITIAL COMPLEX BASED ON THE MECHANISM OF FORMATION OF SUBSEQUENT REACTION-PRODUCTS

The preparation, reactivity patterns, and H-1 NMR and optical spectral properties of the sulfmyoglobin, SMb, complex formed from equine myoglobin reconstituted with deuteroheme are investigated to shed light on the structure of the prosthetic group. The characteristic H-1 NMR spectral properties in both paramagnetic and diamagnetic derivatives of the initially formed green SMb show it to contain an iron chlorin with molecular/electronic structure very similar to that obtained with native protohemin (Chatfield, M. J.; La Mar, G. N.; Kauten, R. J. Biochemistry 1987, 27, 6939-6950), except that the deuterohemin SMb is significantly less stable with respect to reversion to starting material. In low-spin ferric or ferrous derivatives, the initially formed green SMb undergoes a first-order rearrangement that regenerates a red hemin-containing SMb complex distinct from the starting material. In the absence of oxygen, this is a terminal product that slowly precipitates out of solution. In the presence of both oxygen and excess nucleophile such as azide or cyanide, this product reacts further to yield yet another red hemin-containing SMb, which C-13 NMR in the presence of (CN-)-C-13 reveals to have incorporated the anion into the prosthetic group of this terminal reaction product. Using the identity of the perturbed 4-substituents as 4-thiol and 4-thiocyanato in the terminal anaerobic and aerobic SMb reaction products, respectively, as determined by 2D NMR and mass spectrometry of the extracted prosthetic groups (Scharberg, M. A.; La Mar, G. N., following paper), a detailed mechanism for the formation of these two products is presented where this initial episulfide rearranges by hydrogen migration to yield the 4-thiol group. This relatively unstable and reactive group, in turn, reacts with molecular oxygen to give a sulfenic acid for which the hydroxide ion is rapidly replaced by the stronger nucleophile, azide or cyanide. The retention of the incorporated sulfur atom (Berzofsky, J. A.; Peisach, J.; Horrecker, B. L. J. Biol. Chem. 1972, 247, 3783-3791) and its rearrangement to the 3-position of the terminal reaction product of native protohemin-containing Mb (Chatfield, M. J.; La Mar, G. N.; Lecomte, J. T. J.; Balch, A. L.; Smith, K. M.; Langry, K. C. J. Am. Chem. Soc. 1986, 108, 7108-7110), and to the 4-position of the terminal aerobic and anaerobic reaction products of deuteroheme-containing Mb, provide compelling evidence that the initially formed green SMb complex for both native protoheme and synthetic deuteroheme reconstituted protein is an episulfide across the beta-beta bond of pyrrole II.