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Structures of protein-protein complexes involved in electron transfer.

Antonyuk SV, Han C, Eady RR, Hasnain SS - Nature (2013)

Bottom Line: Key to these roles is the formation of transient inter-protein electron transfer complexes.Very high resolution provides the first view of the atomic detail of the interface between the core trimeric cupredoxin structure of CuNiR and the tethered cytochrome c domain that allows the enzyme to function as an effective self-electron transfer system where the donor and acceptor proteins are fused together by genomic acquisition for functional advantage.Comparison of RpNiR with the binary complex of a CuNiR with a donor protein, AxNiR-cytc551 (ref. 6), and mutagenesis studies provide direct evidence for the importance of a hydrogen-bonded water at the interface in electron transfer.

View Article: PubMed Central - PubMed

Affiliation: Molecular Biophysics Group, Institute of Integrative Biology, Faculty of Health and Life Sciences, University of Liverpool, Liverpool L69 7ZX, UK.

ABSTRACT
Electron transfer reactions are essential for life because they underpin oxidative phosphorylation and photosynthesis, processes leading to the generation of ATP, and are involved in many reactions of intermediary metabolism. Key to these roles is the formation of transient inter-protein electron transfer complexes. The structural basis for the control of specificity between partner proteins is lacking because these weak transient complexes have remained largely intractable for crystallographic studies. Inter-protein electron transfer processes are central to all of the key steps of denitrification, an alternative form of respiration in which bacteria reduce nitrate or nitrite to N2 through the gaseous intermediates nitric oxide (NO) and nitrous oxide (N2O) when oxygen concentrations are limiting. The one-electron reduction of nitrite to NO, a precursor to N2O, is performed by either a haem- or copper-containing nitrite reductase (CuNiR) where they receive an electron from redox partner proteins a cupredoxin or a c-type cytochrome. Here we report the structures of the newly characterized three-domain haem-c-Cu nitrite reductase from Ralstonia pickettii (RpNiR) at 1.01 Å resolution and its M92A and P93A mutants. Very high resolution provides the first view of the atomic detail of the interface between the core trimeric cupredoxin structure of CuNiR and the tethered cytochrome c domain that allows the enzyme to function as an effective self-electron transfer system where the donor and acceptor proteins are fused together by genomic acquisition for functional advantage. Comparison of RpNiR with the binary complex of a CuNiR with a donor protein, AxNiR-cytc551 (ref. 6), and mutagenesis studies provide direct evidence for the importance of a hydrogen-bonded water at the interface in electron transfer. The structure also provides an explanation for the preferential binding of nitrite to the reduced copper ion at the active site in RpNiR, in contrast to other CuNiRs where reductive inactivation occurs, preventing substrate binding.

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Details of interactions between cytochrome and Cu binding domains in (a) RpNiR, (b) P93A and (c) M92A in H3 crystal. A 2Fo-Fc map contoured at 1.0 σ at 1.01/1.4/1.8Å resolution for wt/P93A/M92A. For all three structures the distances between Fe and T1 Cu is 10.1-10.3 Å. The interface between cytochrome domain and T1Cu site has 2 conserved water molecules (W1 and W2) located in very close proximity to CBC heme atom (3.5-3.6 and 3.3-3.4Å respectively), the waters are hydrogen bonded to each other. Distances from W1 molecule to His143(Nε2) and Ala138(O) are 2.8Å. Additional water W3 is seen in P93A close to the mutation site.
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Figure 3: Details of interactions between cytochrome and Cu binding domains in (a) RpNiR, (b) P93A and (c) M92A in H3 crystal. A 2Fo-Fc map contoured at 1.0 σ at 1.01/1.4/1.8Å resolution for wt/P93A/M92A. For all three structures the distances between Fe and T1 Cu is 10.1-10.3 Å. The interface between cytochrome domain and T1Cu site has 2 conserved water molecules (W1 and W2) located in very close proximity to CBC heme atom (3.5-3.6 and 3.3-3.4Å respectively), the waters are hydrogen bonded to each other. Distances from W1 molecule to His143(Nε2) and Ala138(O) are 2.8Å. Additional water W3 is seen in P93A close to the mutation site.

Mentions: The potential involvement of Met92 and Pro93 in ET of RpNiR was tested by individual substitution by Ala. Structures of these variants determined at 1.9 Å and 1.4 Å respectively show that the conserved water was not perturbed by these substitutions (Fig.3). The mutations had no significant effect on the specific activity of the enzyme and in single turnover experiments where reduced RpNiR was reoxidised by nitrite, the ET efficiency from heme to T2Cu was similar to the wild-type enzyme, with M92A variant showing a small decrease in rate (see supplementary material). These small effects suggests that the conserved water molecule H-bonded to the solvent-exposed T1Cu histidine ligand and the carbonyl of the peptide bond of Ala138 plays a dominant role in ET in this tethered complex. The nature of this interaction precludes further testing of its involvement in ET by mutation. This role for H-bonded water in ET between the heme and T1Cu site contrasts with the transient binary AxNiR-Cytc551complex where the close contact between the two proteins results in utilization of C-C interactions between the CBC methyl group and Pro88 of the cupredoxin domain of the core NiR. These two ET systems nicely illustrate the two classes of electron tunneling processes predicted ie protein-mediated and structured water–mediated.22


Structures of protein-protein complexes involved in electron transfer.

Antonyuk SV, Han C, Eady RR, Hasnain SS - Nature (2013)

Details of interactions between cytochrome and Cu binding domains in (a) RpNiR, (b) P93A and (c) M92A in H3 crystal. A 2Fo-Fc map contoured at 1.0 σ at 1.01/1.4/1.8Å resolution for wt/P93A/M92A. For all three structures the distances between Fe and T1 Cu is 10.1-10.3 Å. The interface between cytochrome domain and T1Cu site has 2 conserved water molecules (W1 and W2) located in very close proximity to CBC heme atom (3.5-3.6 and 3.3-3.4Å respectively), the waters are hydrogen bonded to each other. Distances from W1 molecule to His143(Nε2) and Ala138(O) are 2.8Å. Additional water W3 is seen in P93A close to the mutation site.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3672994&req=5

Figure 3: Details of interactions between cytochrome and Cu binding domains in (a) RpNiR, (b) P93A and (c) M92A in H3 crystal. A 2Fo-Fc map contoured at 1.0 σ at 1.01/1.4/1.8Å resolution for wt/P93A/M92A. For all three structures the distances between Fe and T1 Cu is 10.1-10.3 Å. The interface between cytochrome domain and T1Cu site has 2 conserved water molecules (W1 and W2) located in very close proximity to CBC heme atom (3.5-3.6 and 3.3-3.4Å respectively), the waters are hydrogen bonded to each other. Distances from W1 molecule to His143(Nε2) and Ala138(O) are 2.8Å. Additional water W3 is seen in P93A close to the mutation site.
Mentions: The potential involvement of Met92 and Pro93 in ET of RpNiR was tested by individual substitution by Ala. Structures of these variants determined at 1.9 Å and 1.4 Å respectively show that the conserved water was not perturbed by these substitutions (Fig.3). The mutations had no significant effect on the specific activity of the enzyme and in single turnover experiments where reduced RpNiR was reoxidised by nitrite, the ET efficiency from heme to T2Cu was similar to the wild-type enzyme, with M92A variant showing a small decrease in rate (see supplementary material). These small effects suggests that the conserved water molecule H-bonded to the solvent-exposed T1Cu histidine ligand and the carbonyl of the peptide bond of Ala138 plays a dominant role in ET in this tethered complex. The nature of this interaction precludes further testing of its involvement in ET by mutation. This role for H-bonded water in ET between the heme and T1Cu site contrasts with the transient binary AxNiR-Cytc551complex where the close contact between the two proteins results in utilization of C-C interactions between the CBC methyl group and Pro88 of the cupredoxin domain of the core NiR. These two ET systems nicely illustrate the two classes of electron tunneling processes predicted ie protein-mediated and structured water–mediated.22

Bottom Line: Key to these roles is the formation of transient inter-protein electron transfer complexes.Very high resolution provides the first view of the atomic detail of the interface between the core trimeric cupredoxin structure of CuNiR and the tethered cytochrome c domain that allows the enzyme to function as an effective self-electron transfer system where the donor and acceptor proteins are fused together by genomic acquisition for functional advantage.Comparison of RpNiR with the binary complex of a CuNiR with a donor protein, AxNiR-cytc551 (ref. 6), and mutagenesis studies provide direct evidence for the importance of a hydrogen-bonded water at the interface in electron transfer.

View Article: PubMed Central - PubMed

Affiliation: Molecular Biophysics Group, Institute of Integrative Biology, Faculty of Health and Life Sciences, University of Liverpool, Liverpool L69 7ZX, UK.

ABSTRACT
Electron transfer reactions are essential for life because they underpin oxidative phosphorylation and photosynthesis, processes leading to the generation of ATP, and are involved in many reactions of intermediary metabolism. Key to these roles is the formation of transient inter-protein electron transfer complexes. The structural basis for the control of specificity between partner proteins is lacking because these weak transient complexes have remained largely intractable for crystallographic studies. Inter-protein electron transfer processes are central to all of the key steps of denitrification, an alternative form of respiration in which bacteria reduce nitrate or nitrite to N2 through the gaseous intermediates nitric oxide (NO) and nitrous oxide (N2O) when oxygen concentrations are limiting. The one-electron reduction of nitrite to NO, a precursor to N2O, is performed by either a haem- or copper-containing nitrite reductase (CuNiR) where they receive an electron from redox partner proteins a cupredoxin or a c-type cytochrome. Here we report the structures of the newly characterized three-domain haem-c-Cu nitrite reductase from Ralstonia pickettii (RpNiR) at 1.01 Å resolution and its M92A and P93A mutants. Very high resolution provides the first view of the atomic detail of the interface between the core trimeric cupredoxin structure of CuNiR and the tethered cytochrome c domain that allows the enzyme to function as an effective self-electron transfer system where the donor and acceptor proteins are fused together by genomic acquisition for functional advantage. Comparison of RpNiR with the binary complex of a CuNiR with a donor protein, AxNiR-cytc551 (ref. 6), and mutagenesis studies provide direct evidence for the importance of a hydrogen-bonded water at the interface in electron transfer. The structure also provides an explanation for the preferential binding of nitrite to the reduced copper ion at the active site in RpNiR, in contrast to other CuNiRs where reductive inactivation occurs, preventing substrate binding.

Show MeSH
Related in: MedlinePlus