Assignment of the ¹³C and ¹³CO resonances for Rhodobacter capsulatus ferrocytochrome c₂ using double‐resonance and triple‐resonance NMR spectroscopy

Abstract

Rhodobacter capsulatus cytochrome c2 uniformly labelled with 13C/15N has been prepared. The 13C resonances of the reduced state, including those of the carbonyl and heme 13C, have been assigned using a combination of various two‐ and three‐dimensional correlated NMR experiments. Assignment of the sidechain 13C resonances facilitated correction of a small number of previously misassigned sidechain 1H and led to the additional assignment of 32 1H. It was found that 13Cα and 13CO secondary shifts were better indicators of secondary structure than 1Hα and 13Cβ secondary shifts. Moreover, it was demonstrated that, despite the significant ring current effects present in heme proteins, 13Cα and 13CO secondary shifts can be employed to accurately identify secondary structure in heme proteins, independently of NOE experiments.

1H and 13C NMR Assignments and Structural Aspects of a Ferrocytochrome c‐551 from the Purple Phototrophic Bacterium Ectothiorhodospira halophila

“…Note that the I3C chemical shift order of the heme methyls is slightly different from previously reported ones. In our case, methyl-2', -7*, -12' and -18' resonate at 13.5, 13.5, 12.8 and 13.7 ppm respectively, in comparison to 13.9, 13.7, 13.5, and 11.5 (horse ferrocytochrome c; Gao et al, 1990), 13.3, 12.4, 11.2, and 9.9 pprn (Rhodobucter cupsulatus ferrocytochrome c,; Caffrey et al, 1994) and to 14.8, 12.8, 12.6 and 12.0 ppm (Desulfovibrio vulgaris ferrocytochrome c-553; Medvedeva et al, 1993). A different chemical shift order of the heme methyl protons may indicate a change in heme ligand geometry, as the chemical shift of these protons is mainly determined by ring current effects in the diamagnetic protein.…”

Mapping the cytochrome c553 interacting site using 1H and 15N NMR

“…This approach may be largely developed for other electron transfer complexes such as cytochrome c /cytochrome c oxidase, cytochrome c /complex bc 1 , etc. However, few cytochromes c have been successfully labelled [18–21]. Cytochrome c overexpression and labelling is a special challenge, mainly for mammalian cytochromes.…”

1H and 13C NMR assignment and secondary structure of Chlorobium limicola f. thiosulfatophilum ferrocytochrome c555

“…4, which have proven to be characteristic of the secondary structure [21]. In diamagnetic heme proteins, the ring current of the porphyrin also contributes to the chemical shift: whereas the IH chemical shift can be significantly biased by this effect (due to the small frequency range for this nucleus (10 ppm)), ~3C NMR results obtained for other cytochromes [5,7,14] have shown that the ring current shift has a minor impact on ~3C chemical shift.…”

“…In our laboratory, we have been studying the structure and dynamic properties of cytochromes c from various species by NMR [5][6][7], in order to understand the underlying reasons for the differences observed in their electron transfer properties. In what follows, we present the assignment of the 1H and ~3C resonances of Chl.…”