The heme group in paramagnetic (S ؍ 1͞2) ferricytochromes c typically displays a markedly asymmetric distribution of unpaired electron spin density among the heme pyrrole  substituents. This asymmetry is determined by the orientations of the heme axial ligands, histidine and methionine. One exception to this is ferricytochrome c552 from Hydrogenobacter thermophilus, which has similar amounts of unpaired electron spin density at the  substituents on all four heme pyrroles. Here, determination of the orientation of the magnetic axes and analysis of NMR line shapes for H. thermophilus ferricytochrome c 552 is performed. These data reveal that the unusual electronic structure for this protein is a result of fluxionality of the heme axial methionine. It is proposed that the ligand undergoes inversion at the pyramidal sulfur, and the rapid interconversion between two diastereomeric forms results in the unusual heme electronic structure. Thus a fluxional process for a metal-bound amino acid side chain has now been identified.
Hydrogenobacter thermophilus cytochrome c(552) ( Ht cyt c(552)) is a small monoheme protein in the cytochrome c(551) family. Ht cyt c(552) is unique because it is hypothesized to undergo spontaneous cytoplasmic maturation (covalent heme attachment) when expressed in Escherichia coli. This is in contrast to the usual maturation route for bacterial cytochromes c that occurs in the cellular periplasm, where maturation factors direct heme attachment. Here, the expression of Ht cyts c(552) in the periplasm as well as the cytoplasm of E. coli is reported. The products are characterized by absorption, circular dichroism, and NMR spectroscopy as well as mass spectrometry, proteolysis, and denaturation studies. The periplasmic product's properties are found to be indistinguishable from those reported for protein isolated from Ht cells, while the major cytoplasmic product exhibits structural anomalies in the region of the N-terminal helix. These anomalies are shown to result from the retention of the N-terminal methionine in the cytoplasmic product, and not from heme attachment errors. The (1)H NMR chemical shifts of the heme methyls of the oxidized ( S=1/2) expression products display a unique pattern not previously reported for a cytochrome c with histidine-methionine axial ligation, although they are consistent with native-like heme ligation. These results support the hypothesis that proper heme attachment can occur spontaneously in the E. coli cytoplasm for Ht cyt c(552).
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