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Lookup NU author(s): Dr Sang-choul Im, Emeritus Prof Alfred Sykes
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The recently reported NMR solution structure of Fe(III)Fe(III) parsley FdI has made possible 2D NOESY NMR studies to determine the point of attachment of Cr(III)L in Fe(III)Fe(III)...Cr(III)L. The latter Cr-modified product was obtained by reduction of Fe(III)Fe(III) parsley and spinach FdI forms with [Cr(15-aneN4) (H2O)2]2+ (15-aneN4 = 1,4,8,12tetraazacyclopentadecane), referred to here as Cr(II)L, followed by air oxidation and chromatographic purification. From a comparison of NMR cross-peak intensities of native and Cr-modified proteins, two surface sites designated A and B, giving large paramagnetic Cr(III)L broadening of a number of amino acid peaks, have been identified. The effects at site A (residues 19-22, 27, and 30) are greater than those at site B (residues 92-94 and 96), which is on the opposite side of the protein. From metal (ICP-AES) and electrospray ionization mass spectrometry (EIMS) analyses on the Cr-modified protein, attachment of a single Cr(III)L only is confirmed for both parsley and spinach FdI and FdII proteins. Electrostatic interaction of the 3+ Cr(III)L center covalently attached to one protein molecule (charge ~-18) with a second (like) molecule provides an explanation for the involvement of two regions. Thus for 3-4 mM Fe(III)Fe(III)...Cr(III)L solutions used in NMR studies (Cr(III)L attached at A), broadening effects due to electrostatic interactions at B on a second molecule are observed. Experiments with the Cys18Ala spinach FdI variant have confirmed that the previously suggested Cys-18 at site A is not the site of Cr(III)L attachment. Line broadening at Val-22 of A gives the largest effect, and Cr(III)L attachment at one or more adjacent (conserved) acidic residues in this region is indicated. The ability of Cr(II)L to bind in some (parsley and spinach) but not all cases (Anabaena variabilis) suggests that intramolecular H-bonding of acidic residues at A is relevant. The parsley and spinach Fe(II)Fe(III)...Cr(III)L products undergo a second stage of reduction with the formation of Fe(II)Fe(II)...Cr(III)L. However, the spinach Glu92Ala (site B) variant undergoes only the first stage of reduction, and it appears that Glu-92 is required for the second stage of reduction to occur. A sample of Cr(III)L-modified parsley Fe(III)Fe(III) Fd is fully active as an electron carrier in the NADPH-cytochrome c reductase reaction catalyzed by ferredoxin-NADP+ reductase.
Author(s): Im S-C, Worrall JAR, Liu G, Aliverti A, Zanetti G, Luchinat C, Bertini I, Sykes AG
Publication type: Article
Publication status: Published
Journal: Inorganic Chemistry
Year: 2000
Volume: 39
Issue: 8
Pages: 1755-1764
ISSN (print): 0020-1669
ISSN (electronic): 1520-510X
Publisher: American Chemical Society
URL: http://dx.doi.org/10.1021/ic991127w
DOI: 10.1021/ic991127w
PubMed id: 12526565
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