Proton conduction within the reaction centers of Rhodobacter capsulatus: The electrostatic role of the protein

P. Maróti, Deborah K. Hanson, Laura Baciou, Marianne Schiffer, Pierre Sebban

Research output: Contribution to journalArticle

58 Citations (Scopus)

Abstract

Light-induced charge separation in the photosynthetic reaction center results in delivery of two electrons and two protons to the terminal quinone acceptor Q(B). In this paper, we have used flash-induced absorbance spectroscopy to study three strains that share identical amino acid sequences in the Q(B) binding site, all of which lack the protonatable amino acids Glu- L212 and Asp-L213. These strains are the photosynthetically incompetent site- specific mutant Glu-L212/Asp-L213 → Ala-L212/Ala-L213 and two different photocompetent derivatives that carry both alanine substitutions and an intergenic suppressor mutation located far from Q(B) (class 3 strain, Ala- Ala + Arg-M231 → Leu; class 4 strain, Ala-Ala + Asn-M43 → Asp). At pH 8 in the double mutant, we observe a concomitant decrease of nearly 4 orders of magnitude in the rate constants of second electron and proton transfer to Q(n) compared to the wild type. Surprisingly, these rates are increased to about the same extent in both types of suppressor strains but remain >2 orders of magnitude smaller than those of the wild type. In the double mutant, at pH 8, the loss of Asp-L213 and Glu-L212 leads to a substantial stabilization (≥60 meV) of the semiquinone energy level. Both types of compensatory mutations partially restore, to nearly the same level, the original free energy difference for electron transfer from primary quinone Q(A) to Q(B). The pH dependence of the electron and proton transfer processes in the double-mutant and the suppressor strains suggests that when reaction centers of the double mutant are shifted to lower pH (1.5-2 units), they function like those of the suppressor strains at physiological pH. Our data suggest that the main effect of the compensatory mutations is to partially restore the negative electrostatic environment of Q(B) and to increase an apparent 'functional' pK of the system for efficient proton transfer to the active site. This emphasizes the role of the protein in tuning the electrostatic environment of its cofactors and highlights the possible long- range electrostatic effects.

Original languageEnglish
Pages (from-to)5617-5621
Number of pages5
JournalProceedings of the National Academy of Sciences of the United States of America
Volume91
Issue number12
DOIs
Publication statusPublished - Jun 7 1994

Fingerprint

Rhodobacter capsulatus
Static Electricity
Protons
Electrons
Proteins
Photosynthetic Reaction Center Complex Proteins
Genetic Suppression
Mutation
Viperidae
Alanine
Amino Acid Sequence
Catalytic Domain
Spectrum Analysis
Binding Sites
Light
Amino Acids

Keywords

  • electron transfer
  • photochemical reaction center
  • proton transfer
  • site- specific mutagenesis

ASJC Scopus subject areas

  • Genetics
  • General

Cite this

Proton conduction within the reaction centers of Rhodobacter capsulatus : The electrostatic role of the protein. / Maróti, P.; Hanson, Deborah K.; Baciou, Laura; Schiffer, Marianne; Sebban, Pierre.

In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 91, No. 12, 07.06.1994, p. 5617-5621.

Research output: Contribution to journalArticle

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abstract = "Light-induced charge separation in the photosynthetic reaction center results in delivery of two electrons and two protons to the terminal quinone acceptor Q(B). In this paper, we have used flash-induced absorbance spectroscopy to study three strains that share identical amino acid sequences in the Q(B) binding site, all of which lack the protonatable amino acids Glu- L212 and Asp-L213. These strains are the photosynthetically incompetent site- specific mutant Glu-L212/Asp-L213 → Ala-L212/Ala-L213 and two different photocompetent derivatives that carry both alanine substitutions and an intergenic suppressor mutation located far from Q(B) (class 3 strain, Ala- Ala + Arg-M231 → Leu; class 4 strain, Ala-Ala + Asn-M43 → Asp). At pH 8 in the double mutant, we observe a concomitant decrease of nearly 4 orders of magnitude in the rate constants of second electron and proton transfer to Q(n) compared to the wild type. Surprisingly, these rates are increased to about the same extent in both types of suppressor strains but remain >2 orders of magnitude smaller than those of the wild type. In the double mutant, at pH 8, the loss of Asp-L213 and Glu-L212 leads to a substantial stabilization (≥60 meV) of the semiquinone energy level. Both types of compensatory mutations partially restore, to nearly the same level, the original free energy difference for electron transfer from primary quinone Q(A) to Q(B). The pH dependence of the electron and proton transfer processes in the double-mutant and the suppressor strains suggests that when reaction centers of the double mutant are shifted to lower pH (1.5-2 units), they function like those of the suppressor strains at physiological pH. Our data suggest that the main effect of the compensatory mutations is to partially restore the negative electrostatic environment of Q(B) and to increase an apparent 'functional' pK of the system for efficient proton transfer to the active site. This emphasizes the role of the protein in tuning the electrostatic environment of its cofactors and highlights the possible long- range electrostatic effects.",
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AU - Maróti, P.

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AU - Sebban, Pierre

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N2 - Light-induced charge separation in the photosynthetic reaction center results in delivery of two electrons and two protons to the terminal quinone acceptor Q(B). In this paper, we have used flash-induced absorbance spectroscopy to study three strains that share identical amino acid sequences in the Q(B) binding site, all of which lack the protonatable amino acids Glu- L212 and Asp-L213. These strains are the photosynthetically incompetent site- specific mutant Glu-L212/Asp-L213 → Ala-L212/Ala-L213 and two different photocompetent derivatives that carry both alanine substitutions and an intergenic suppressor mutation located far from Q(B) (class 3 strain, Ala- Ala + Arg-M231 → Leu; class 4 strain, Ala-Ala + Asn-M43 → Asp). At pH 8 in the double mutant, we observe a concomitant decrease of nearly 4 orders of magnitude in the rate constants of second electron and proton transfer to Q(n) compared to the wild type. Surprisingly, these rates are increased to about the same extent in both types of suppressor strains but remain >2 orders of magnitude smaller than those of the wild type. In the double mutant, at pH 8, the loss of Asp-L213 and Glu-L212 leads to a substantial stabilization (≥60 meV) of the semiquinone energy level. Both types of compensatory mutations partially restore, to nearly the same level, the original free energy difference for electron transfer from primary quinone Q(A) to Q(B). The pH dependence of the electron and proton transfer processes in the double-mutant and the suppressor strains suggests that when reaction centers of the double mutant are shifted to lower pH (1.5-2 units), they function like those of the suppressor strains at physiological pH. Our data suggest that the main effect of the compensatory mutations is to partially restore the negative electrostatic environment of Q(B) and to increase an apparent 'functional' pK of the system for efficient proton transfer to the active site. This emphasizes the role of the protein in tuning the electrostatic environment of its cofactors and highlights the possible long- range electrostatic effects.

AB - Light-induced charge separation in the photosynthetic reaction center results in delivery of two electrons and two protons to the terminal quinone acceptor Q(B). In this paper, we have used flash-induced absorbance spectroscopy to study three strains that share identical amino acid sequences in the Q(B) binding site, all of which lack the protonatable amino acids Glu- L212 and Asp-L213. These strains are the photosynthetically incompetent site- specific mutant Glu-L212/Asp-L213 → Ala-L212/Ala-L213 and two different photocompetent derivatives that carry both alanine substitutions and an intergenic suppressor mutation located far from Q(B) (class 3 strain, Ala- Ala + Arg-M231 → Leu; class 4 strain, Ala-Ala + Asn-M43 → Asp). At pH 8 in the double mutant, we observe a concomitant decrease of nearly 4 orders of magnitude in the rate constants of second electron and proton transfer to Q(n) compared to the wild type. Surprisingly, these rates are increased to about the same extent in both types of suppressor strains but remain >2 orders of magnitude smaller than those of the wild type. In the double mutant, at pH 8, the loss of Asp-L213 and Glu-L212 leads to a substantial stabilization (≥60 meV) of the semiquinone energy level. Both types of compensatory mutations partially restore, to nearly the same level, the original free energy difference for electron transfer from primary quinone Q(A) to Q(B). The pH dependence of the electron and proton transfer processes in the double-mutant and the suppressor strains suggests that when reaction centers of the double mutant are shifted to lower pH (1.5-2 units), they function like those of the suppressor strains at physiological pH. Our data suggest that the main effect of the compensatory mutations is to partially restore the negative electrostatic environment of Q(B) and to increase an apparent 'functional' pK of the system for efficient proton transfer to the active site. This emphasizes the role of the protein in tuning the electrostatic environment of its cofactors and highlights the possible long- range electrostatic effects.

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KW - site- specific mutagenesis

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