Comparison of oxidation-reduction site geometries in oxidized and reduced Chromatium high potential iron protein and oxidized Peptococcus aerogenes ferredoxin.

Abstract We have calculated a difference Fourier map between oxidized and reduced Chromatium high potential iron protein (HiPIP). The most conspicuous features of this map show that the reduced iron-sulfur (Fe4S*4) cluster shrinks slightly upon oxidation, in agreement with results of independent least squares refinement of cluster atom parameters in both oxidation states. The simplest interpretation of this change is that the cluster contracts about 0.16 A in the direction of four Fe-S* bonds lying approximately perpendicular to the plane of the intramolecular interface (Carter, C. W., Jr., Kraut, J., Freer, S. T., Xuong, Ng. H., Alden, R. A., and Bartsch, R. G. (1974) J. Biol. Chem, 249, 4212–4225). A smaller contraction of about 0.08 A probably occurs along the direction of a second set of four Fe-S* bonds, and a very slight expansion, about 0.03 A, may occur in the remaining direction. A tentative conclusion is that the oxidized cluster is more nearly of tetrahedral symmetry than is the reduced cluster. This change may also be accompanied by slight rearrangements of the nonpolar side chains surrounding the cluster, notably at Tyr 19, but no significant conformational change appears to occur in the backbone. The cluster is bound in a large interior cavity by covalent Fe-Sγ links to cysteine residues 43, 46, 63, and 77. This cavity is constructed from residues 43 to 80, and consists primarily of hydrogen-bonded secondary structures, including several hairpin turns and five strands of twisted antiparallel β sheet. These secondary structures also provide three backbone N-H to Sγ hydrogen bonds to the cluster-linked sulfur atoms of cysteine residues 46, 63, and 77. The immediate environment of the Fe4S*4 cluster is completed by a number of aromatic and other nonpolar side chains which interpenetrate this hydrogen-bonded framework. Solvent is apparently excluded from contact with the cluster by this arrangement. Sequence comparisons among three related high potential iron proteins (Chromatium, Dus, K., Tedro, S., and Bartsch, R. G. (1973) J. Biol. Chem. 248, 7318–7331; Thiocapsa pfennigii, Tedro, S. M., Meyer, T. E., and Kamen, M. D. (1974) J. Biol. Chem. 249, 1182–1188; and Rhodopseudomonas gelatinosa, Tedro, S., personal communication) suggest that these features of the cluster binding cavity are preserved in all three species. We can partly rationalize the structural changes that occur upon oxidation by the orientation of the cluster in this cavity. These observations are consistent with an earlier proposal that the Fe4S*4 cluster functions differently in HiPIP than it does in bacterial ferredoxin (Carter, C. W., Jr, Kraut, J., Freer, S. T., Alden, R. A., Sieker, L. C., Adman, E., and Jensen, L. H. (1972) Proc. Nat. Acad. Sci. U. S. A. 69, 3526–3529). The aromatic ring of Tyr 19 is in contact with the Fe4S*4 cluster in Chromatium HiPIP. A similar relationship between tyrosine side chains and chromophores has been found to occur in Peptococcus aerogenes ferredoxin (Adman, E. T., Sieker, L. C., and Jensen, L. H. (1973) J. Biol. Chem. 248, 3987–3996) and two bacterial flavodoxins (Watenpaugh, K. D., Sieker, L. C., Jensen, L. H., Legall, j., and Du- Bourdieu, M. (1972) Proc. Nat. Acad. Sci. U. S. A. 69, 3185–3188; Anderson, R. D., Apgar, P. A., Burnett, R. M., Darling, G. D., Lequesne, M. E., Mayhew, S. G., and Ludwig, M. L. (1972) Proc. Nat. Acad. Sci. U. S. A. 69, 3189–3191). These observations, together with 13C nuclear magnetic resonance measurements showing that this relationship also coccurs in Clostridium acidi-urici ferredoxin (Packer, E. L., Sternlicht, H., and Rabinowitz, J. C. (1972) Proc. Nat. Acad. Sci. U. S. A. 69, 3278–3282), strongly suggest that Tyr 19 plays an active role in the oxidation-reduction mechanism of Chromatium HiPIP.