J. Biol. Chem., Vol. 266, Issue 35, 23714-23723, Dec, 1991
Two-dimensional NMR investigation of iron-sulfur cluster electronic and molecular structure of oxidized Clostridium pasteurianum ferredoxin. Interpretability of contact shifts in terms of cysteine orientation
SC Busse, GN La Mar and JB Howard
Department of Chemistry, University of California, Davis 95616.
A two-dimensional NMR study has been carried out on the four-iron clusters
of a bacterial oxidized ferredoxin for the purpose of investigating the
relationship between contact shift patterns and the orientation of the
individual coordinated cysteines. The ferredoxin from Clostridium
pasteurianum, CpFdox, was selected because of its extensive sequence
homology, and likely close structural similarity, to the
crystallographically characterized ferredoxin from Peptococcus aerogenes,
Pa Fdox (Adman, E.T., Sieker, L.C., and Jensen, L. H. (1973) J. Biol. Chem.
248, 3987-3996). Rapid data collection rates with minimal but adequate
acquisition time allowed the detection of numerous CpFdox cross-peaks from
the contact-shifted and strongly relaxed coordinated cysteinyl C beta H
protons in the resolved 10-20 ppm window. Relatively strong magnitude COSY
cross peaks from the resolved eight cysteinyl C beta H resonance
unambiguously locate the geminal C beta H partner for each residue; weaker
cross-peaks locate the C alpha Hs from three of the residues. The geminal
nature of the magnitude-COSY detected partners to the resolved C beta H
peaks is confirmed by strong NOESY cross-peaks. The NOESY spectra,
moreover, assign an additional two cysteinyl C alpha H resonances. The
present results confirm some previous one-dimensional NOE assignments,
revise others, and locate resonances previously undetected (Bertini, I.,
Briganti, F., Luchinat, C., and Scozzafara, A. (1990) Inorg. Chem. 29,
1874-1880). A striking pairwise pseudo-symmetry in cysteinyl contact shift
patterns is observed which is attributed to the previously recognized
pseudo- symmetry in the crystal of PaFdox. A detailed analysis of the
structural/electronic determinants of the coordinated cysteine C beta H
contact shift pattern is made, and the NMR data necessary for unique
interpretation are identified. It is shown that analysis of the relaxation
properties of cysteine beta-methylene protons provides the stereospecific
assignments necessary for comparison of shift ratios with crystallographic
structural data. The available structural data on PaFdox (Backes, G., Mino,
Y., Loehr, T., Meyer, T., Cusanovich, M., Sweeney, W., Adman, E., and
Sanders-Loehr, J. (1991) J. Am. Chem. Soc. 13, 2055-2064) are qualitatively
but not quantitatively consistent with the observed cysteinyl contact shift
pattern, with the NMR data reflecting more asymmetry than previous studies.
A tentative assignment of a single pair of symmetry-related cysteines is
proposed.(ABSTRACT TRUNCATED AT 400 WORDS)
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