The heme iron in cytochrome c-550 from Thiobacillus versutus has a methionine and a histidine as axial ligands. In order to study the characteristics of a possible lysine-histidine ligation in a heme protein, the methionine has been replaced by a lysine. This residue acts as a ligand between pH 3 and 12. The midpoint potential of the mutant has shifted -329 mV compared to wild type, but apart from this shift the pH dependence of the midpoint potential is unchanged, suggesting that the large drop is caused by specific ligand effects and not by protein refolding. While the EPR spectrum of wild-type cytochrome c-550 shows one species with gz = 3.35, in the spectrum of the mutant two species occur with gz values of 3.53 and 3.30. The intensity ratio of both species depends on the presence of organic cosolvents. In the low frequency region (-4 to -1 ppm) of the 1H NMR spectrum of mutant ferrocytochrome c-550, four one-proton peaks replace the resonances of the ligand methionine side chain protons. Using two-dimensional NMR spectroscopy (COSY and NOESY), these protons and five others have been assigned to the lysine ligand. The spectroscopic results obtained for this mutant show similarities with those observed for the alkaline form of cytochrome c, supporting the Lys-His ligation proposed for this protein. The data are consistent with the evidence for amine ligation in cytochrome f: the EPR spectrum of M100K cytc-550 is similar to that of cytochrome f. However, the NMR spectra show significant differences.(ABSTRACT TRUNCATED AT 250 WORDS)

A soluble monoheme c-type cytochrome (cytochrome c,) has been isolated from the green alga Monoraphidium braunii. It has a molecular mass of 9.3 kDa, an isoelectric point of 3.6 and a reduction potential of 358 mV at pH 7. The determined amino acid sequence allows its classification as a class-I c-type cytochrome. The femc and ferrous cytochrome forms and their pH equilibria have been studied using 'H-NMR, ultraviolet/visible, EPR and Mossbauer spectroscopies. The pH equilibria are complex, several pK, values and pH-dependent forms being observed. The amino acid sequence, the reduction-potential value and the visible and NMR spectroscopies data in the pH range 4-9 indicate that the heme iron has a methionine-histidine axial coordination. However, the EPR and Mossbauer data obtained for the ferricytochrome show that in this pH range two distinct forms are present: form I, g, = 3.27, g, = 2.05 and g, = 1.05; form 11, g, = 2.95, g, = 2.29 and g, = 1.43. While form I has crystal-field parameters typical of a methionine-histidine coordination, those associated with form I1 would suggest a histidine-histidine axial ligation. This possibility was extensively analyzed by spectroscopic methods and by chemical modification of a histidine residue. It was concluded that form I1 actually corresponds to an unusual type of methionine-histidine axial coordination. Straightforward examples of this type of coordination have recently been found in other c-type hemeproteins [

A seven-iron ferredoxin was isolated from aerobically grown cells of the hyperthermoacidophilic archaleon Desulfurolobus ambivalens (DSM 3772). The protein is monomeric, with an apparent molecular mass of 15 kDa and contains 7 iron atoms/molecule. The N-terminal sequence shows a large similarity (70% identity) with that of the ferredoxin isolated from the archaeon Sulfolobus acidocaldarius. The EPR isharacteristics in both the native (oxidized) and dithionite-reduced states of this protein allowed an unequivocal identification of a [3Fe-4SI1+" center, with a reduction potential of -270-+ 20 mV, at pH 7.5. The protein also contains a [4Fe-4S]*'"+ center with a very low reduction potential (Eo = -540 mV, pH 7.1D), which yields a rhombic EPR spectrum upon reduction with sodium dithionite at high pH. The reduction potentials of both centers are slightly pH dependent between pH 6 and 9. The [3Fe-4S] ferredoxin center is able to accept electrons from pyruvate oxidase and NADH oxidase isolated from D. ambivalens. This ferredoxin is present in large amounts (at least 130 mgkg wet cells), which allowed the unequivocal observation of oxidized [3Fe-4S] clusters in intact D. ambivalens cells.Keywords. Iron-sulfur centers ; Archaea; EPR; thermophilic.Proteins containing iron-sulfur clusters with a wide range of stoichiometries are ubiquitously found to be involved in fundamental biological processes like nitrogen fixation, CO, fixation, hydrogen metabolism, the citric-acid cycle, detoxification and membrane-bound (energy-transduction processes [l -31. The simplest iron-sulfur proteins, the ferredoxins, are involved in these pathways as one-electron carriers. Due mainly to the extensive work on model compounds, it has been shown that under anaerobic conditions iron-sulfur structures may self-assemble in reaction mixtures containing iron, sulfide and thiols [4]. Thus, since iron-sulfur proteins are present in the three major urkingdoms, they were proposed as the most ancient electron-transfer agents to have appeared during biological evolution [5]. Therefore, in evolutionary terms it is particularly interesting to screen ffor iron-sulfur proteins in archaea, the most ancient living organisms [6].

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