Proton NMR characterization of isomeric sulfmyoglobins: preparation, interconversion, reactivity patterns, and structural features

Abstract

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.

Three-dimensional structure of cyanomet-sulfmyoglobin C.

“…Horse heart ScMb-CN was prepared from horse heart metmyoglobin (metMb) (Sigma) as described by Chatfield et al (29) for preparation of ScMb from sperm whale Mb. Bright green crystals of ScMb were grown following the published protocol for the native protein (30).…”

Biogenesis of reactive sulfur species for signaling by hydrogen sulfide oxidation pathways

“…Sulfide can also react with ferrous-oxy heme, leading, in a series of steps, to sulfur addition to the porphyrin ring, forming sulfhemoglobin 27 . The latter is an irreversible modification also seen with myoglobin 28 and is therefore unlikely to be useful in a signaling context.…”

NMR and EPR Studies of Chloroiron(III) Tetraphenyl-chlorin and Its Complexes with Imidazoles and Pyridines of Widely Differing Basicities

“…There has previously been some confusion regarding the classification of reduced hemes and the expectations as to their electronic properties, and a number of careful structural investigations were required to elucidate the geometric and stereoisomeric structures of these macrocycles. [1][2][3][4][5][6][7][8][9][10][11][12] There have been a number of previous studies of synthetic and natural iron chlorin and/or isobacteriochlorin complexes by magnetic resonance techniques, including the seminal paper of Stolzenberg, Strauss and Holm in 1981 13 and additional papers from these authors, 14-18 as well as other researchers. [19][20][21][22][23][24][25][26][27][28][29][30][31] Understanding the electronic properties of the iron(III) complexes of each of the individual "green" hemes is an important step in understanding their mechanisms of action.…”