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The enzymes can exist as monomers or homodimers of the core structure, as well as heterodimers with an additional cytochrome c subunit [10] , [12] — [16]. The SOR from Campylobacter jejuni , though originally defined as a two-subunit protein acting similarly to the S. With the exception of the enzyme from Deinococcus radiodurans , all characterized SORs were interestingly found to be encoded upstream their putative physiological electron acceptors c -type cytochromes or other redox proteins [19].

Consistently, the genes coding for the c -type cytochromes identified as electron acceptors for SORs from S. These c -type cytochromes are also very diverse. They differ in size and heme content and this is an additional feature contributing to the complexity of SORs. Several attempts have been made to elucidate how SOR-mediated sulfite oxidation is integrated in cell metabolism.

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It is postulated that Group 1 SORs are directly linked to the respiratory chain via their natural electron acceptor cytochromes [20]. Similarly, in C. Although such a scenario seems plausible, also in this case the exact electron transfer pathway and the redox proteins involved have not been identified.

Only cell extracts, and not purified proteins, were used in these experiments and, indeed, one cannot exclude the involvement of additional, unidentified electron shuttles. In the former study [20] , however, experiments were performed using isolated cell membranes and the association of the electron acceptor of SorT, cytochrome c Smc, with cytochrome oxidases remains to be proven.

On the contrary, in the latter study on T. Accordingly, the electrons generated upon sulfite oxidation by SOR TTHB8 are transferred to the natural electron acceptor of the enzyme, cytochrome c , and from here to cytochrome c , the physiological electron donor of the two terminal cytochrome c oxidases, ba 3 and caa 3. Here, the role of T. Native ba 3 - and caa 3 -type cytochrome c oxidases were isolated from T.

Native cytochrome c was purified according to Soulimane and co-authors [23]. Expression and purification of the proteins were conducted as described previously [16].

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The flowthrough was collected, the column washed with the equilibration buffer and finally bound proteins were eluted with the same buffer containing mM NaCl. The sequence encoding the mature N-terminal domain of cytochrome c c 2076 was amplified from T. These constructs permit the expression of the recombinant domains in E. Periplasmic proteins were prepared from fresh biomass.


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The supernatant containing the recombinant domain was extensively dialyzed against 5 mM Tris-HCl pH 8. The C-terminal domain was expressed and initially extracted as described above for the N-terminal domain. The supernatant containing the c [C] domain was extensively dialyzed against 5 mM Tris-acetate buffer pH 6. The last step was repeated twice. Analysis of the association of cytochrome c domains was carried out by analytical size exclusion chromatography SEC. Elution profiles were recorded at nm. The cytochrome full-length was analysed in a similar way.


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Stopped-flow experiments were carried out with a thermostated instrument DX. Reactions were investigated by monitoring the absorption changes at selected wavelengths. To prevent inhibition of SOR TTHB8 resulting from prolonged incubation of the enzyme with a large excess of sulfite, in these experiments the stopped-flow instrument was used in the sequential mixing mode.

The reduction of the cytochrome or its domains was monitored at nm or, in the case of a too high signal in the Soret region, at nm. This low temperature was chosen to slow-down and thus better resolve in time the reactions. Experiments were carried out in 5 mM Bis-Tris pH 7. Observed rate constants k obs were therefore obtained by fitting the experimental time courses to the equations described in [26] for the analysis of bimolecular reactions assayed under second-order conditions.

Approximately 1. The reaction was followed at nm. The recently identified periplasmic di-heme cytochrome c [16] as a whole shows poor similarity to known proteins. However, when analyzed separately, its N-terminal domain sequence containing one heme binding site presents a high homology to the subunit B of SOR from C. This led to the hypothesis that cytochrome c is likely organized in two distinct domains, each one possibly presenting an independent fold and a heme cofactor, with distinct roles in mediating electron transfer between SOR TTHB8 and the respiratory chain, through cytochrome c [16].

Consistently, according to the DomPred server [27] cytochrome c consists of two domains, with a predicted boundary at residue and a proline rich region 91— aa likely representing a flexible inter-domain linker [28] Figure 1A. While globular proteins, due to their native rigid structure, are typically resistant to proteolysis under physiological conditions, flexible inter-domain linkers can be substrates for proteases.

Cytochromes C: Evolutionary, Structural and Physicochemical Aspects

This makes the limited proteolysis approach suitable to confirm the multi-domain organization of a protein [29]. Based on this notion, cytochrome c was subjected to limited proteolysis using trypsin. Overall, the cytochrome presents 19 putative cleavage sites for this protease Figure 1A. Among these sites, the one in position K is of particular interest, as it is located within the predicted linker, being therefore potentially more accessible to the protease. On this basis, limited proteolysis of the recombinant cytochrome c is expected to yield the N- and C-terminal domains as the major cleavage products.

The schematic representation of the full-length cytochrome c FL with predicted trypsin cleavage sites vertical lines and the cloned c 2076 and c [C] domains is shown in panel A.


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The predicted boundary of the two domains is indicated by an arrow. Lanes: 1— molecular marker; 2— full-length cytochrome c ; 3— protein sample in the column flowthrough assigned to c [C]; 4— protein sample eluting from the column at mM NaCl assigned to c 2076.

As c 2076 and c [C] have significantly different calculated isoelectric points 5 and 7. As expected, two major products of limited proteolysis were obtained in addition to the band corresponding to non-digested cytochrome Figure 1B. The bigger fragment in the flowthrough can be attributed to c [C], while the shorter fragment eluting at mM NaCl likely corresponds to c 2076. This result confirms that the protein as a whole is strongly dipolar with a negatively charged c 2076 and a positively charged c [C].

This is most likely due to the presence of multiple trypsin cleavage sites within the protein, especially those located at the termini of the domains, more easily accessed by the protease Figure 1A. The limited proteolysis did not disturb the core of the domains or the heme binding, as the two generated fragments exhibited UV-Vis spectroscopic properties identical to those of the individually expressed domains see below.

This indicates that the two proteolytic fragments correspond to the two domains , each of them being independently folded and associated with one heme cofactor. The use of recombinantly produced, isolated polypeptidic domains has proven to be a valuable approach to investigate intra- and intermolecular electron transfer between redox centers in multi-domain proteins with largely overlapping spectral properties [30] , [31].

Cytochromes C: Evolutionary, Structural and Physicochemical Aspects

Therefore, to further confirm the existence of two independent domains in cytochrome c and to investigate their function, the N- and C-terminal parts of the protein were individually produced in E. The recombinant domains contained the PelB signal sequence to promote their translocation to the periplasmic space and fragments of the flexible linker in order to enhance the stability of both domains Figure 1A.

After protein expression and isolation of the periplasmic fractions, the two domains were purified by IEX and size exclusion chromatography SEC , as described in the Materials and Methods section. This shows that the splitting of the signal does not arise from the presence of two hemes. Instead, it is the result of the transition between the ground state and two or more excited states close in energy. Overall, the spectral properties confirmed that one heme cofactor was successfully incorporated in each domain during recombinant expression in E.

Lanes: 1 and 3— purified c [C] and c 2076, respectively; 2— molecular marker. Inset: enlargement of the visible region. Analytical SEC chromatograms relative to from top to bottom the full-length cytochrome c , a mixture of c [C] and c 2076, and each of the two domains analyzed separately. Separation by IEX chromatography of the domains obtained by limited proteolysis of cytochrome c Fig.

All together the results presented above indicate that cytochrome c folds into two independent domains with distinct properties, each carrying a single heme group. These features may have implications with regard to the electron transfer activity of the cytochrome. We have previously shown that cytochrome c is the physiological electron acceptor of the sulfite:cytochrome c oxidoreductase encoded by the ttha gene in T. Based on the high similarity between c 2076 and the SorB subunit of the sulfite:cytochrome c oxidoreductase from C.

Similarly, a positively charged surface area surrounding the pocket cradling the molybdopterin cofactor is present also in the SOR from T. Structure analysis of SOR TTHB8 revealed the positively charged surface area of the protein A, encircled , believed to be important for interaction with cytochrome c The hydrophobic belt surrounding the heme cleft in cytochromes c B and c C is depicted in red.

In order to test which domain of cytochrome c preferably interacts with SOR TTHB8 , the electron transfer between the latter enzyme and the recombinant c 2076 or c [C] was kinetically investigated by stopped-flow spectroscopy. This implies that c 2076 and c [C] are in rapid redox equilibrium and can exchange electrons once reduction of the c 2076 has occurred. Under all conditions a linear concentration dependence was observed.

Interestingly, c 2076, either isolated or integrated in the full-length protein, is reduced at comparable rates, which further points to the N-terminal domain of cytochrome c as the electron acceptor for SOR TTHB8. Panel C shows the turnover rates TN for c red , c 2076 blue and a mixture of c 2076 and c [C] green , calculated as described in Materials and Methods.

We have previously shown that cytochrome c passes the electrons generated during sulfite oxidation to the terminal oxidases of the respiratory chain via cytochrome c To assess which domain of cytochrome c takes part in this electron transfer, the oxidation of c 2076 or c [C] by cytochrome c was tested. The reaction was studied under non pseudo-first order conditions and, accordingly, the experimental traces were fitted following the analysis described elsewhere [26]. From the results presented in Figure 5AB it can be concluded that, despite the low temperature, both c 2076 and c [C] are able to rapidly exchange electrons with cytochrome c Table 1 shows the estimated forward k F and reverse k R rate constants of the reaction measured at the same ionic strength 12 mM.

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The observed differences in k F and k R are not significant due to the rather high experimental error in those measurements, partly arising from the large optical overlap among the investigated proteins. Based on the results, we conclude that both c 2076 and c [C] exchange electrons with cytochrome c at rates similar to those previously measured with the full-length cytochrome c [16]. Interestingly, the reaction between c 2076 and c displays a similar ionic strength dependence to the full-length protein, whereas the dependence is less pronounced in the case of c [C] Figure 5C. This suggests that electrostatic forces play an important role in the interaction between c 2076 and c , while apolar interactions may be involved in molecular recognition between c [C] and c To investigate the electron transfer between c 2076, c [C] and c , kinetic traces were collected after anaerobically mixing 3.

Panel C shows the ionic strength dependence of the reaction of c red , c 2076 blue or c [C] black with c The heme cleft in the very well described cytochrome c is surrounded by a hydrophobic belt Figure 3C consisting of residues G13, C14, F26, V68, M69 and F72; this patch of residues likely participates in the interaction between the cytochrome and the terminal oxidase ba 3 [36]. Interestingly, the model structure of c [C] clearly shows that such hydrophobic residues G11, C12, F24, V66, M67 and F70 are structurally conserved around the cleft Figure 3B.

It is therefore likely that molecular recognition between c [C] and c is also mediated by this hydrophobic patch, in line with the modest ionic strength dependence reported in Figure 5C. When assayed separately, c 2076, similarly to full-length c , is very slowly oxidized by either ba 3 or caa 3 oxidase, whereas c [C] is quickly oxidized by either of the two oxidases Figure 6A,B.

Considering the high sequence similarity between c [C] and c and the structurally conserved hydrophobic belt Figure 3BC likely participating in molecular recognition between c and ba 3 -oxidase, this finding is perhaps not unexpected. On the other hand, we have previously shown and confirmed here that the electron transfer between full-length c and ba 3 or caa 3 is not efficient, unless mediated by c acting as an electron shuttle [16]. Therefore, we suggest that the relatively fast direct electron transfer between c [C] and terminal oxidases here documented is of no physiological value.

If the conserved hydrophobic patch mentioned above is involved in mediating electron transfer from c [C], in the full-length protein such a patch should be not accessible by large molecules, like ba 3 or caa 3 oxidase, though possibly still allowing interaction with much smaller molecules such as cytochrome c In C and D, the oxidation of c 2076 or c [C] by ba 3 oxidase was investigated in the presence of 10 nM c magenta.

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Expectedly, in the presence of 10 nM oxidized c , both c 2076 and c [C] are promptly oxidized by ba 3 Figure 6C,D or caa 3 data not shown , as previously observed with the full-length protein [16]. This further supports a fast electron transfer between either of the two domains of cytochrome c and cytochrome c While sulfite oxidizing enzymes SOEs in vertebrates and humans have been studied for over 40 years, significant progress in studying bacterial SOEs has only been made in the last decade. Bacterial SOEs are extremely diverse in terms of structure and oxidizing proteins.

Discovery of novel SOEs is being constantly reported, but in most cases the relative electron acceptor was not identified. Hence, usually the link to the respiratory chain is not shown [12] — [14] , [18] , [20].