Cleavage of the iron-methionine bond in c-type cytochromes: Crystal structure of oxidized and reduced cytochrome c2 from Rhodopseudomonas palustris and its ammonia complex

Abstract

The three-dimensional structures of the native cytochrome c 2 from Rhodopseudomonas palustris and of its ammonia complex have been obtained at pH 4.4 and pH 8.5, respectively. The structure of the native form has been refined in the oxidized state at 1.70 Å and in the reduced state at 1.95 Å resolution. These are the first high-resolution crystal structures in both oxidation states of a cytochrome c 2 with relatively high redox potential (+350 mV). The differences between the two oxidation states of the native form, including the position of internal water molecules, are small. The unusual six-residue insertion Gly82-Ala87, which precedes the heme binding Met93, forms an isolated 3 10 -helix secondary structural element not previously observed in other c-type cytochromes. Furthermore, this cytochrome shows an external methionine residue involved in a strained folding near the exposed edge of the heme. The structural comparison of the present cytochrome c 2 with other c-type cytochromes has revealed that the presence of such a residue, with torsion angles and of approximately −140 and −130°, respectively, is a typical feature of this family of proteins. The refined crystal structure of the ammonia complex, obtained at 1.15 Å resolution, shows that the sulphur atom of the Met93 axial ligand does not coordinate the heme iron atom, but is replaced by an exogenous ammonia molecule. This is the only example so far reported of an X-ray structure with the heme iron coordinated by an ammonia molecule. The detachment of Met93 is accompanied by a very localized change in backbone conformation, involving mainly the residues Lys92, Met93, and Thr94. Previous studies under typical denaturing conditions, including high-pH values and the presence of exogenous ligands, have shown that the detachment of the Met axial ligand is a basic step in the folding/unfolding process of c-type cytochromes. The ammonia adduct represents a structural model for this important step of the unfolding pathway. Factors proposed to be important for the methionine dissociation are the strength of the H-bond between the Met93 and Tyr66 residues that stabilizes the native form, and the presence in this bacterial cytochrome c 2 of the rare six-residue insertion in the helix 3 10 conformation that increases Met loop flexibility.

The effect of replacing the axial methionine ligand with a lysine residue in cytochrome c‐550 from Paracoccus versutus assessed by X‐ray crystallography and unfolding

“…The refined M100K structure confirmed previous spectroscopic evidence that the Lys side chain is buried in the protein interior with the amino group coordinating to the ferric heme-iron [7]. The K100 N firon distance of 1.9 Å is considerably shorter than the Fe-N distance in Fe-NH 3 model complexes (2.1 Å ) and in the cyt c 2 ammonia adduct (2.1 Å ) [24]. Furthermore, the distance is longer for Lys heme-iron coordination in the octaheme tetrathionate reductase from Shewanella oneidensis MR-1, 2.2 Å [47], and also for the N-terminal amino coordination in cytochromes f, 2.1 Å [48].…”

“…4C). The changes in this region are not as large as those observed in the imidazole or ammonia cyt c 2 adducts [23,24] which show peptide bond flipping and 180° rotation of side chains. This is most likely due to the fact that in the latter the absence of coordination by the Met does not impose any conformational constraints and the localized region of the loop affected by release of the Met ligand is then free to adopt a new low energy conformation.…”

“…Cyt c-550, on the other hand, is a low-potential cyt c 2 with a reduction potential of +250 mV vs. NHE, pH 7.0 [3]. The recent structures of a high-potential cyt c 2 (E ¼ +350-450 mV vs. NHE) in both oxidation states have given insight into a possible explanation for their elevated reduction potentials [24,44], assigned to a lack of movement of a highly conserved water molecule upon oxidation of the heme to the Fe 3+ state. This water is present as wat5 in the cyt c-550 structure (Fig.…”

“…For exogenous ligands to gain access to the heme, sufficient movement of the loop containing the Met ligand connecting helices four and five must occur [19–22]. At present two X‐ray structures of cyts c 2 exist in which the coordinating Met‐iron bond is broken and the vacant heme coordination site is filled by an exogenous ligand; the imidazole adduct of Rhodobacter sphaeroides cyt c 2 [23] and the ammonia adduct of Rhodopseudomonas palustris cyt c 2 [24]. In both structures loss of Met ligation does not result in wholesale structural changes but is localized to a few residues adjacent to the now noncoordinating Met residue.…”

Exploring structurally conserved solvent sites in protein families

“…In addition, our method identified two novel sites with 93 and 86% conservation (Table I, sites 1 and 3). A water molecule located in site 1 has been discussed separately for cytochrome c 2 from Rhodopila globiformis ,33 Rhodopseudomonas palustris ,34 and Paracoccus denitrificans 35. To our knowledge, the wider conservation of this site among monodomain cytochrome c structures has not been previously appreciated.…”