Bacterial Cytochromes C2 Research Articles

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

AbstractThe three‐dimensional structures of the native cytochrome c2 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 c2 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 310‐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 c2 with other c‐type cytochromes has revealed that the presence of such a residue, with torsion angles ϕ and ξ 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 c2 of the rare six‐residue insertion in the helix 310 conformation that increases Met loop flexibility.

Redox chemistry and acid-base equilibria of mitochondrial plant cytochromes c.

Mitochondrial cytochromes c from spinach, cucumber, and sweet potato have been investigated through direct electrochemical measurements and electronic and 1H NMR spectroscopies, under conditions of varying temperature and pH. The solution behaviors of these plant cytochromes closely resemble, but do not fully reproduce, those of homologous eukaryotic species. The reduction potentials (E0') at pH 7 and 25 degrees C are +0.268 V (spinach), +0.271 V (cucumber), and +0.274 V (sweet potato) vs SHE. Three acid-base equilibria have been determined for the oxidized proteins with apparent pKa values of 2.5, 4.8, and 8.3-8.9, which are related to disruption of axial heme ligation, deprotonation of the solvent-exposed heme propionate-7 and replacement of the methionine axially bound to the heme iron with a stronger ligand, respectively. The most significant peculiarities with respect to the mammalian analogues include: (i) less negative reduction enthalpies and entropies (Delta S0'rc and Delta H0'rc) for the various protein conformers [low- and high-T native (N1 and N2) and alkaline (A)], whose effects at pH 7 and 25 degrees C largely compensate to produce E degrees ' values very similar to those of the mammalian proteins; (ii) the N1 --> N2 transition that occurs at a lower temperature (e.g., 30-35 degrees C vs 50 degrees C at pH 7. 5) and at a lower pH (7 vs 7.5); and (iii) a more pronounced temperature-induced decrease in the pKa for the alkaline transition which allows observation of the alkaline conformer(s) at pH values as low as 7 upon increasing the temperature above 40 degrees C. Regarding the pH and the temperature ranges of existence of the various protein conformers, these plant cytochromes c are closer to bacterial cytochromes c2.

The molecular structure of an unusual cytochrome c2 determined at 2.0 A; the cytochrome cH from Methylobacterium extorquens.

Cytochrome cH is the electron donor to the oxidase in methylotrophic bacteria. Its amino acid sequence suggests that it is a typical Class 1 cytochrome c, but some features of the sequence indicated that its structure might be of special interest. The structure of oxidized cytochrome cH has been solved to 2.0 A resolution by X-ray diffraction. It has the classical tertiary structure of the Class 1 cytochromes c but bears a closer gross resemblance to mitochondrial cytochrome c than to the bacterial cytochrome c2. The left-hand side of the haem cleft is unique; in particular, it is highly hydrophobic, the usual water is absent, and the "conserved" Tyr67 is replaced by tryptophan. A number of features of the structure demonstrate that the usual hydrogen bonding network involving water in the haem channel is not essential and that other mechanisms may exist for modulation of redox potentials in this cytochrome.

Interaction of horse cytochromec with the photosynthetic reaction center ofRhodospirillum rubrum

Mitochondrial cytochrome c (horse), which is a very efficient electron donor to bacterial photosynthetic reaction centers in vitro, binds to the reaction center of Rhodospirillum rubrum with an approximate dissociation constant of 0.3-0.5 microM at pH 8.2 and low ionic strength. The binding site for the reaction center is on the frontside of cytochrome c which is the side with the exposed heme edge, as revealed by differential chemical acetylation of lysines of free and reaction-center-bound cytochrome c. In contrast, bacterial cytochrome c2 was found previously to bind to the detergent-solubilized reaction center through its backside, i.e., the side opposite to the heme cleft [Rieder, R., Wiemken, V., Bachofen, R., and Bosshard, H. R. (1985). Biochem. Biophys. Res. Commun. 128, 120-126]. Binding of mitochondrial cytochrome c but not of mitochondrial cytochrome c2 is strongly inhibited by low concentrations of poly-L-lysine. The results are difficult to reconcile with the existence of an electron transfer site on the backside of cytochrome c2.

Characterisation of ionisations that influence the redox potential of mitochondrial cytochrome c and photosynthetic bacterial cytochromes c2

Several cytochromes c 2 from the Rhodospirillaceae show a pH dependence of redox potential in the physiological pH range which can be described by equations involving an ionisation in the oxidised form (p K o) and one in the reduced form (p K r). These cytochromes fall into one of two groups according to the degree of separation of p K o and p K r. In group A, represented here by the Rhodomicrobium vannielii cytochrome c 2, the separation is approx. one pH unit and the ionisation is that of a haem propionic acid. Members of this group are unique among both cytochromes c 2 and mitochondrial cytochromes c in lacking the conserved residue Arg-38. We propose that the role of Arg-38 is to lower the p K of the nearby propionic acid, so that it lies out of the physiological pH range. Substitution of this residue by an uncharged amino acid leads to a raised p K for the propionic acid. In group B, represented here by Rhodopseudomonas viridis cytochrome c 2, the separation between p K o and p K r is approx. 0.4 pH unit and the ionisable group is a histidine at position 39. This was established by NMR spectroscopy and confirmed by chemical modification. Only a few other members of the cytochrome c 2/mitochondrial cytochrome c family have a histidine at this position and of these, both Crithidia cytochrome c-557 and yeast cytochrome c were found to have a pH-dependent redox potential similar to that of Rps. viridis cytochrome c 2. Using Coulomb's law, it was found that the energy required to separate p K o and p K r could be accounted for by simple electrostatic interactions between the haem iron and the ionisable group.

Electron transfer reactions of high-potential iron-sulfur proteins and c-type cytochromes.

Studies of electron transfer by biological oxidation-reduction proteins frequently focus on the interaction of a particular protein with nonphysiological oxidants and/or reductants. This approach, although valuable, is limited by the size and chemistry of the nonphysiological reactants. To further the understanding of biological electron transfer, we have investigated the interaction of two examples of high-potential iron-sulfur proteins (HIPIP's) with mitochondrial cytochrome c (horse heart) and bacterial cytochrome c2 from Rhodospirillum rubrum, Rhodopseudomonas palustris, Rhodopseudomonas capsulata, and Rhodopseudomonas sphaeroides. On the basis of the kinetics of electron transfer between the various HIPIP's and cytochromes, it appears that the interactions are more complex than those observed with nonphysiological reactants. We conclude that (1) specific sites on both the HIPIP's and the cytochromes mediate electron transfer with the effect of ionic strength different from that expected on the basis of the interaction of the various proteins with iron hexacyanides, (2) the interaction of HIPIP with some of the cytochromes investigated is heterogeneous, resulting from at least two possible orientations (cytochrome dependent) for interaction leading to electron transfer, and (3) no long-lived complexes between the HIPIP's and cytochromes are formed due to rapid equilibrium between the two proteins. This last conclusion suggests that the measured second-order rate constant is in fact the product of the association constant (for any HIPIP and a particular cytochrome) and a first-order rate constant reflecting the rate-limiting step leading to products.

Redox potentials of the photosynthetic bacterial cytochromes c2 and the structural bases for variability

The cytochromes c 2 of the Rhodospirillaceae show a much greater variation in redox potential and its pH dependence than the mitochondrial cytochromes c that have been studied. It is proposed that the range of redox potential for cytochromes c 2 functioning as the immediate electron donor to photo-oxidised bacteriochlorophyll may be 345–395 mV at pH 5. Closely related cytochromes c 2 with different redox potentials show patterns of amino acid substitution which are consistent with changes in hydrophobicity near the haem being at least a partial determinant of redox potential. More distantly related cytochromes are difficult to compare because of the large number of amino acid substitutions and the probability that there are subtle changes in overall peptide chain folding. The redox potential versus pH curves can be analysed in terms of either one ionisation in the oxidised form or two in the oxidised form and one in the reduced. The p K in the oxidised form at higher pH values can be correlated with the p K for the disappearance or shift of the near infrared absorption band located near 695 nm. The structural bases of these ionisations are not known but the possible involvement of the haem propionate residues is discussed.

Reduction kinetics of bacterial cytochromes c2

In a continuing effort to understand the mechanism of electron transfer by c-type cytochromes we have extended our investigations of the oxidation and reduction of Rhodospirillum rubrum cytochrome c 2. We have utilized the oxidant, oxidized azurin, and the reductants SO 2 −, S 2O 4 2−, sodium ascorbate, and reduced azurin. The results of these studies demonstrate that, as found previously with the iron hexacyanides, electron transfer apparently takes place at the exposed heme edge. Furthermore, we report studies on the reduction of ferricytochrome c 2 from Rhodopseudomonas sphaeroides, Rhodopseudomonas capsulata, Rhodomicrobium vannielii, and Rhodopseudomonas palustris by potassium ferrocyanide. Based on the amino acid sequence homology between the various cytochromes c 2 and presumed structural homology, the observed rates of electron transport are analyzed in terms of the structure in the region of the exposed heme edge.