Proton-assisted Two-electron Transfer in Natural Variants of Tetraheme Cytochromes from Desulfomicrobium Sp.

Abstract

The tetraheme cytochrome c3 isolated from Desulfomicrobium baculatum (DSM 1743)(Dsmb) was cloned, and the sequence analysis showed that this cytochrome differs in just three amino acid residues from the cytochrome c3 isolated from Desulfomicrobium norvegicum (Dsmn): (DsmnXXDsmb) Thr-37 → Ser, Val-45 → Ala, and Phe-88 → Tyr. X-ray crystallography was used to determine the structure of cytochrome c3 from Dsmb, showing that it is very similar to the published structure of cytochrome c3 from Dsmn. A detailed thermodynamic and kinetic characterization of these two tetraheme cytochromes c3 was performed by using NMR and visible spectroscopy. The results obtained show that the network of cooperativities between the redox and protonic centers is consistent with a synergetic process to stimulate the hydrogen uptake activity of hydrogenase. This is achieved by increasing the affinity of the cytochrome for protons through binding electrons and, reciprocally, by favoring a concerted two-electron transfer assisted by the binding of proton(s). The data were analyzed within the framework of the differences in the primary and tertiary structures of the two proteins, showing that residue 88, close to heme I, is the main cause for the differences in the microscopic thermodynamic parameters obtained for these two cytochromes c3. This comparison reveals how replacement of a single amino acid can tune the functional properties of energy-transducing proteins, so that they can be optimized to suit the bioenergetic constraints of specific habitats. The tetraheme cytochrome c3 isolated from Desulfomicrobium baculatum (DSM 1743)(Dsmb) was cloned, and the sequence analysis showed that this cytochrome differs in just three amino acid residues from the cytochrome c3 isolated from Desulfomicrobium norvegicum (Dsmn): (DsmnXXDsmb) Thr-37 → Ser, Val-45 → Ala, and Phe-88 → Tyr. X-ray crystallography was used to determine the structure of cytochrome c3 from Dsmb, showing that it is very similar to the published structure of cytochrome c3 from Dsmn. A detailed thermodynamic and kinetic characterization of these two tetraheme cytochromes c3 was performed by using NMR and visible spectroscopy. The results obtained show that the network of cooperativities between the redox and protonic centers is consistent with a synergetic process to stimulate the hydrogen uptake activity of hydrogenase. This is achieved by increasing the affinity of the cytochrome for protons through binding electrons and, reciprocally, by favoring a concerted two-electron transfer assisted by the binding of proton(s). The data were analyzed within the framework of the differences in the primary and tertiary structures of the two proteins, showing that residue 88, close to heme I, is the main cause for the differences in the microscopic thermodynamic parameters obtained for these two cytochromes c3. This comparison reveals how replacement of a single amino acid can tune the functional properties of energy-transducing proteins, so that they can be optimized to suit the bioenergetic constraints of specific habitats. A variety of mechanisms used by nature to perform coupled reactions, a fundamental requirement for energy transduction processes, can now be established in detail for relatively small macromolecules that can couple the transfer of electrons and protons. The tetraheme cytochrome c3 is found in all organisms belonging to the Desulfovibrionaceae family as well as in other sulfate-reducing bacteria including Desulfomicrobium (Dsm) species, where it is produced in large quantities (1Coutinho I.B. Xavier A.V. Methods Enzymol. 1994; 243: 119-140Crossref PubMed Scopus (71) Google Scholar). It is a soluble protein of low molecular weight, with the c-type hemes covalently bound to the polypeptide chain by two thioether bridges provided by cysteine residues arranged in the typical -CXX(XX)CH-heme binding sequences, and axially coordinated by two histidines. Recently, tetraheme cytochromes c3 have been classified as type I and type II (2Valente F.M. Saraiva L.M. LeGall J. Xavier A.V. Teixeira M. Pereira I.A. Chembiochem. 2001; 2: 895-905Crossref PubMed Scopus (57) Google Scholar), and the Dsm cytochromes studied in this work have mixed characteristics of both types. Like type I, these cytochromes have a heme-binding site of type -CXXXXCH-, acidic residues around heme I, and long N- and C-terminal regions. However, the region of positive charges surrounding heme IV, which is characteristic of type I cytochromes c3, is not well defined in these cytochromes. The close proximity of the hemes in cytochrome c3 results in homotropic redox cooperativities. Furthermore, their reduction potentials very and in the LeGall J. Xavier A.V. J. PubMed Scopus Google LeGall J. Xavier A.V. J. PubMed Scopus Google Scholar). these properties cytochromes c3 to couple the transfer of electrons and protons A.V. J. Scopus Google Scholar), which for type I cytochrome c3 (2Valente F.M. Saraiva L.M. LeGall J. Xavier A.V. Teixeira M. Pereira I.A. Chembiochem. 2001; 2: 895-905Crossref PubMed Scopus (57) Google is fundamental for as of the M. PubMed Scopus Google LeGall J. Xavier A.V. J. 2: Scopus (57) Google Scholar). The Desulfomicrobium norvegicum cytochrome c3 been to be in transfer between the and the cytochrome c3 M. M. PubMed Scopus Google in a similar as that for type I and type II cytochromes c3 from (2Valente F.M. Saraiva L.M. LeGall J. Xavier A.V. Teixeira M. Pereira I.A. Chembiochem. 2001; 2: 895-905Crossref PubMed Scopus (57) Google Scholar). the of the residues in homotropic cooperativities in type I cytochromes c3 from LeGall J. Xavier A.V. J. PubMed Scopus Google and J. M. J. PubMed Scopus Google LeGall J. Xavier A.V. J. PubMed Scopus Google Scholar). were used to the of specific residues in the of the redox properties of these L.M. LeGall J. Xavier A.V. J. Scopus Google L.M. LeGall J. Xavier A.V. PubMed Scopus Google Saraiva L.M. Xavier A.V. 2001; PubMed Scopus Google Scholar). these cytochromes homotropic between two hemes a coordinated two-electron a positive their characterization of the cytochrome c3 from LeGall J. Xavier A.V. Chembiochem. 2001; 2: PubMed Scopus Google Xavier A.V. J. 2001; PubMed Scopus Google to a coordinated two-electron transfer positive homotropic redox cooperativities. This is achieved by the concerted of two positive a coordinated two-electron Furthermore, these positive for the activity of this cytochrome LeGall J. Xavier A.V. PubMed Scopus Google Scholar). cytochromes c3 from Dsm were isolated from organisms that in habitats. norvegicum J. Scopus Google was isolated in a from a from J. PubMed Scopus Google Scholar), and Desulfomicrobium baculatum (DSM was isolated from the of the and Scopus Google as of a with a PubMed Scopus Google Scholar). However, these two have J. Scopus Google Scholar), a very close with this these Dsm cytochromes c3 have a similar primary with and Phe-88 in the cytochrome c3 from by Ser, Ala, and in the cytochrome c3 from as in this by is to that of M. M. J. 1994; 243: PubMed Scopus Google Scholar). as be from the in their microscopic thermodynamic properties The of similar proteins, as the of cytochromes c3 from and Dsmb, the of in the primary sequence that with the of it to the to the of the protein that cytochrome c3 from M. M. M. J. J. PubMed Scopus Google not of cytochrome c3 from Dsmb, which was in a between and using as of cytochrome c3 from were obtained using the were obtained the was by the which the and of the c3 from in the with parameters a and with in the and a of data were a the using a of The were and by and and structure were with the 1994; PubMed Scopus Google Scholar). The of the data in of data in to the in to the in to the in to the in to the in a and structure of cytochrome c3 from was by molecular replacement using the J. 1994; Scopus Google with the structure of the cytochrome c3 from M. M. J. 1994; 243: PubMed Scopus Google as a A with a of and of was The including and a of were performed with A for and using the molecular replacement and data to The that was A of was using Methods Enzymol. PubMed Scopus Google using a of a PubMed Scopus Google was from of the and were and a using J. and to chain to the to for chain and to to the The for including a were that the be visible both the and and be hydrogen and The heme were The main chain of residues and were not so was and used for the a of this the was to and the to The large between the two the of in the NMR data that Phe-88 was by a I.B. LeGall J. Xavier A.V. J. PubMed Scopus Google Scholar), the of the cytochrome c3 which was the process was showed two Thr-37 and were in the and with PubMed Scopus Google Scholar). This as a for the all the The in The and structures have been the with the protein from of where is the for the from the is the of the from the and is the of for this which the of where is the for the from the is the of the from the and is the of for this which the in a were used for F.M. F.M. in Scholar). The sequence of the residues of cytochrome c3 was found to be to the residues of cytochrome c3 from Dsmn. the amino acid sequence and that the C-terminal of the two cytochromes two were and using these a sequence from the The was and of were both with the and analyzed c3 from and were as (1Coutinho I.B. Xavier A.V. Methods Enzymol. 1994; 243: 119-140Crossref PubMed Scopus (71) Google Scholar), and the NMR were a of The of the was to through the of a of in to the transfer The of the was for by small of of and The were not for the J. Scopus Google and the of in and in The and of the protein were obtained by the LeGall J. Xavier A.V. PubMed Scopus (71) Google Scholar), using of the isolated from as NMR NMR were obtained a with a with and a The of the was The were with a of and a of both The were with data using a in and in were using as with the of by redox of both cytochromes were performed using of the LeGall J. Xavier A.V. Chembiochem. 2001; 2: PubMed Scopus Google using protein in acid and The protein the of with to the The redox were to the defined in the Methods Enzymol. PubMed Scopus Google and of the of c3. Scholar). 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J. 1994; Scopus Google Scholar). with the transfer from the the of electrons the protein is and it can be to reduction 2001; 2: PubMed Scopus Google Scholar). the of the kinetic the reduction potentials of the redox centers and their The was used to the The of the NMR were used as of the of The of the in the visible and kinetic were to have of and to the thermodynamic parameters and the the for the in the and and the of the visible were optimized LeGall J. Xavier A.V. Chembiochem. 2001; 2: PubMed Scopus Google Scholar). 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J. 1994; 243: PubMed Scopus Google and these residues have a in the redox and properties of of the the residue that is a in the cytochrome from and a in the cytochrome from Dsmb, is in the of the protein close to heme The reduction potentials of the hemes in cytochrome c3 from of with heme I the with differences around for the other three hemes and The reduction potentials in be a of the of the of the hemes in the of the which the reduction of this cytochrome The of close to heme I is with the for the reduction of this The all very similar in the two as the of between the two The redox and centers positive cooperativities well with the of the with heme in both cytochromes. The in this is to a of the of the to of data low is in comparison the of the the with heme this not the main of as heme this heme I the of all the with the protonic in both This of heme I a for the as for other cytochromes c3 LeGall J. Xavier A.V. J. PubMed Scopus Google L.M. LeGall J. Xavier A.V. 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This very well with the of the in The in is and hemes I, and heme through and transfer to a that hemes II and a to heme the of a with heme II the hemes I and in a reduction of the with the coordinated transfer of two which in cytochrome c3 from to a of the of of these two hemes in the both have positive and IV, it is that the affinity for protons of the cytochrome which is for the activity of low LeGall J. Xavier A.V. J. 2: Scopus (57) Google Scholar). However, and the of the positive cooperativities is not to the of protons. the of results in a coordinated transfer of two the electrons to a transfer and the protonic of the cytochrome is protons that can be to LeGall J. Xavier A.V. PubMed Scopus Google A.V. PubMed Scopus Google A.V. PubMed Scopus Google Scholar), transfer of electrons and protons that to the energy-transducing However, this of the of typical and transfer be from the thermodynamic properties of the isolated the reduction potentials of the isolated cytochrome close to a coordinated two-electron transfer that a in by the can the to it It is to that this small to a of to both the protons and the electrons from the of is for a be through small in the a of other and the two-electron transfer as in cytochrome c3 from LeGall J. Xavier A.V. Chembiochem. 2001; 2: PubMed Scopus Google Xavier A.V. J. 2001; PubMed Scopus Google Scholar). It is that the in thermodynamic parameters that the is the functional be to the functional to how this is in the thermodynamic parameters obtained for these two Dsm cytochromes that they of energy transduction using a from that LeGall J. Xavier A.V. J. PubMed Scopus Google LeGall J. Xavier A.V. PubMed Scopus Google Xavier A.V. J. PubMed Scopus Google LeGall J. Xavier A.V. 1994; PubMed Scopus Google LeGall J. Xavier A.V. J. Scopus Google for and type I cytochromes c3. The in the microscopic thermodynamic parameters of these two by a small in their amino acid and have from with the from which these two organisms were was obtained as a and this is the of the the other was isolated from a between bacteria and sulfate-reducing bacteria PubMed Scopus Google Scholar). the primary of the process is a very low redox The of a by the a of the thermodynamic properties of in the bioenergetic for the to the from which the was the the of for the and and for the used for were by and were by the for the of the in the