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ELECTRON CARRIERS AND B12 TRANSPORTER PROTEINS
Gianpiero Garau, Dipartimento di Scienze Chimiche, Università di Trieste.
Cytochromes c2 are biological electron shuttles involved in cyclic redox processes . They are structurally and evolutionarily correlated to mitochondrial cyt c. The midpoint redox potential of all eukaryotic cyt c is about +260 mV, while it varies from +260 to +450 mV in bacterial proteins. The hydrophobic heme pocket of c-type cyts is of interest to investigate factors controlling the midpoint redox potential and the folding/unfolding pathway of these proteins. In eukaryotic cyts, the positional change of a buried water molecule, located in the heme binding pocket and conserved in almost all the X-ray structures, is the most important aspect observed comparing the oxidized and reduced form. A similar positional change was reported in 1996 for the bacterial Blastochloris viridis cyt c2. This presents a reduction potential of +285 mV, so close to that of eukaryotic proteins. We have determined the X-ray structures of the oxidized and reduced form of the cyt c2 from Rodopseudomonas palustris that has a reduction potential of +365 mV.1, 2 These are the first high resolution crystal structures of a c-type cyt with a high redox potential, obtained in both redox states. In all oxidized forms of c-type cyts with low reduction potential the buried water molecule is found in a position different from that in the reduced forms. On the contrary, in the present cyt this water molecule is detected in a position close to that of all reduced forms (Fig. 1).
A rough evaluation of the electrostatic interactions between the buried water molecule and its H-bond amino acids points out that in c-type cyts with low reduction potential the movement of the water molecule lowers by about 100 mV the reduction potential through the stabilisation of the oxidised form. These findings confirm that the buried water molecule plays an important role in adjusting the redox potential of c-type cyts, with alternation of the surrounding H-bond network.3 The small movement of the buried water molecule in R. palustris cyt c2 can be associated to the H-bond distance between Met axial ligand and Tyr residue in the heme binding pocket. The increase of this distance in R. palustris cyt c2 with respect eukaryotic cyts could "disconnect the communication" between the metal centre and the water molecule. As a consequence, the water molecule is less influenced by the change of the iron oxidation state.3
The detachment of the Met axial ligand from iron co-ordination is the first step of c-type cytochrome unfolding pathway, observed in solution upon increasing pH, T or using antagonist exogenous ligands. We have obtained the ammonia complex structure of R. palustris cytochrome c2 at atomic resolution and this structure shows that the detachment of the Met axial ligand is accompanied by a very localised change in the backbone conformation, involving mainly Lys92, Met93 and Thr 94 (Fig. 2).1 With respect to the native form, the Lys92 backbone is flipped and this forces the Met loop to turn away from the core of the protein. The ammonia complex structure represents a model for the first step of the c-type cyt unfolding process.
The importance of B12 -transporter proteins is clearly evidenced by the role of cobalamins (Cbls) in cellular metabolic processes. Vitamin B12 is synthesised only by certain micro-organism and mammalians are ultimately dependent on its external sources. Assimilation of the dietary B12 is a complex process with three Cbl-trasporters, haptocorrin (HC), intrinsic factor (IF) and transcobalamin (TC), as well as several specific membrane receptors. Any failure in the internalisation process causes B12 deficiency that leads to abnormalities such as megaloblastic anaemia and disorder of the nervous system. Rapidly proliferating tissues, such as those of several tumours, are highly susceptible to the uptake of Cbls. The three proteins consist of a core of approximately 400 residues, which is highly glycosylated for IF and HC but not for TC. This feature makes TC particularly suitable for crystallisation. Alignment of TC amino acid sequences with IF and HC shows clusters of high similarity with sixt regions where the homology is close to 80%. This suggest a common ancestral gene for the three transporters. Determination of the ternary structure of a member of B12 transporters is essential to obtain a better insight into the adsorption, transportation and cellular uptake of B12 derivatives (Fig. 3).
Human TC was expressed and purified at Science Park laboratories of Aarhus University (Dk). Crystals of human Trascobalamin suitable for X-ray analysis were obtained and a complete data set was collected to 3.2 Å resolution at Elettra.4 Systematic absences were in agreement with a space group P212121. The self rotation peaks suggest the presence of a non-crystallographic two-fold symmetry in the [011] direction. Attempts to improve the resolution and to search for heavy-atom derivatives are under way.
1. Geremia S., Garau G., Vaccari L., Sgarra R., Viezzoli M. S., Calligaris M. and Randaccio L. (2001). 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 Protein Sci. 11, 6-17.
2. Garau G., Geremia S., Vaccari L., Viezzoli M. S. and Randaccio L. (2000). Crystallization and preliminary X-ray analysis of two pH-dependent forms of cytochrome c2 from Rhodopseudomonas palustris Acta Cryst. D56, 1699-1701.
3. Garau G., Geremia S., Randaccio L. (2002). Relationship between hydrogen-bonding network and reduction potential in c-type cytochromes. Febs Lett., submitted.
4. Garau G., Fedosov S. V., Petersen T., Geremia S. and Randaccio L. (2001) Crystallization and preliminary X-ray diffraction analysis of human Transcobalamin, a vitamin B12-transporting protein Acta Cryst. D57, 1890-1892.
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