The (CrL)-L-II reduction of [2Fe-2S] ferredoxins and site of attachment ofCr-III using H-1 NMR and site-directed mutagenesis

The recently reported NMR solution structure of (FeFeIII)-Fe-III parsley Fd I has made possible 2D NOESY NMR studies to determine the point of attachme nt of (CrL)-L-III in (FeFeIII)-Fe-III...(CL)-L-III. The latter Cr modified product was obtained by reduction of (FeFeIII)-Fe-III parsley and spinach F dI forms with [Cr(15-aneN(4)) (H2O)(2)](2+) (15-aneN(4) = 1,4,8,12-tetraaza cyclopentadecane), referred to here as (CrL)-L-II, followed by air oxidatio n and chromatographic purification. From a comparison of NMR cross-peak int ensities of native and Cr-modified proteins, two surface sites designated A and B, giving large paramagnetic (CrL)-L-III broadening of a number of ami no 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), whic h is on the opposite side of the protein. From metal (ICP-AES) and electros pray ionization mass spectrometry (EIMS) analyses on the Cr-modified protei n, attachment of a single (CrL)-L-III only is confirmed for both parsley an d spinach FdI and FdII proteins. Electrostatic interaction of the 3+ (CrL)- L-III center covalently attached to one protein molecule (charge similar to -18) with a second (like) molecule provides an explanation for the involvem ent of two regions. Thus for 3-4 mM (FeFeIII)-Fe-III...(CrL)-L-III solution s used in NMR studies ((CrL)-L-III attached at A), broadening effects due t o electrostatic interactions at B on a second molecule are observed. Experi ments with the Cys18Ala spinach FdI variant have confirmed that the previou sly suggested Cys-18 at site A is not the site of (CrL)-L-III attachment. L ine broadening at Val-22 of A gives the largest effect, and (CrL)-L-III att achment at one or more adjacent (conserved) acidic residues in this region is indicated. The ability of (CrL)-L-II to bind in some (parsley and spinac h) but not all cases (Anabaena variabilis) suggests that intramolecular H-b onding of acidic residues at A is relevant. The parsley and spinach (FeFeII I)-Fe-II...(CrL)-L-III products undergo a second stage of reduction with th e formation of (FeFeII)-Fe-II...(CrL)-L-III. However, the spinach Glu92Ala (site B) variant undergoes only the first stage of reduction, and it appear s that Glu-92 is required for the second stage of reduction to occur. A sam ple of (CrL)-L-III-modified parsley (FeFeIII)-Fe-III Fd is fully active as an electron carrier in the NADPH-cytochrome c reductase reaction catalyzed by ferredoxin-NADP(+) reductase.