The preparations of sulfmyoglobin (sulf-Mb) by standard procedures have been found heterogeneous by 1H NMR spectroscopy. Presented here are the results of a comprehensive study of the factors that influence the selection among the three dominant isomeric forms of sperm whale sulf-Mb and their resulting detailed optical and 1H NMR properties as related to their detectability and structural properties of the heme pocket. A single isomer is formed initially in the deoxy state; further treatment in any desired oxidation/ligation state can yield two other major isomers. Acid catalysis and chromatography facilitate formation of a second isomer, particularly in the high-spin state. At neutral pH, a third isomer is formed by a first-order process. The processes that alter oxidation/ligation state are found to be reversible and are judged to affect only the metal center, but the three isomeric sulf-Mbs are found to exhibit significantly different ligand affinity and chemical stability. The present results allow, for the first time, a rational approach for preparing a given isomeric sulf-Mb in an optimally pure state for subsequent characterization by other techniques. While optical spectroscopy can distinguish the alkaline forms, only 1H NMR clearly distinguishes all three ferric isomers. The ring current shifts in the carbonyl complexes of reduced sulf-Mb complexes support saturation for a pyrrole in each isomer. The hyperfine shift patterns in the various oxidation/spin states of sulf-Mbs indicate relatively small structural alteration, and the proximal and distal sides of the heme suggest that peripheral electronic effects are responsible for the differentially reduced ligand affinities for the three isomeric sulf-Mbs. The first 1H NMR spectra of sulfhemoglobins are presented, which indicate a structure similar to that of the initially formed sulf-Mb isomer but also suggest the presence of a similar molecular heterogeneity as found for sulf-Mb, albeit to a smaller extent.

No abstract

No abstract

No abstract

Analysis of the 1H NMR hyperfine shift patterns of isomeric sulfmyoglobins is carried out in the met-aquo and met-cyano states to determine the site of saturation in each protein. The utility of the patterns for structure elucidation is established by specific deuterium labeling of the heme methyls of the terminal base product. On the basis of the known saturation of ring B in this isomer [Chatfield, M.J., La Mar, G.N., Lecomte, J.T.J., Balch, A.L., Smith, K.M., & Langry, K.C. (1986) J. Am. Chem. Soc. 108, 7108-7110], the methyl resonance of the saturated ring is found to have strongly attenuated contact shift. Thus, the heme methyl contact shift pattern is diagnostic for the saturated pyrrole in the high-spin state. This rationale is then applied to analyze the assigned NMR spectra of the initial and terminal acid sulfmyoglobin products, revealing that the same ring B is saturated in each isomer. In contrast, the heme methyl contact shift pattern in low-spin ferric complexes reveals that the methyls both on the affected pyrrole and on the trans pyrrole are influenced similarly on sulfmyoglobin formation, precluding the use of this methyl shift pattern as a unique indicator of the site of saturation. Identification of exchangeable proximal histidine resonances for met-aquo sulfmyoglobin complexes with shifts similar to that in native myoglobin dictates inconsequential axial alterations in the sulfmyoglobins, while location of downfield meso proton resonances analogous to those of the native protein demonstrates the retention of the coordinate water in the active site of met-sulfmyoglobin.