Fourier transform infrared and mass spectrometry analyses of a site-directed mutant of D1-Asp170 as a ligand to the water-oxidizing Mn4CaO5 cluster in photosystem II

Tomomi Kitajima-Ihara, Takehiro Suzuki, Shin Nakamura, Yuichiro Shimada, Ryo Nagao, Naoshi Dohmae, Takumi Noguchi

Research output: Contribution to journalArticle

2 Citations (Scopus)

Abstract

The Mn4CaO5 cluster, the catalytic center of water oxidation in photosystem II (PSII), is coordinated by six carboxylate and one imidazole ligands. The roles of these ligands in the water oxidation mechanism remain largely unknown. In this study, we constructed a D1-D170H mutant, in which the Asp ligand bridging Mn and Ca ions was replaced with His, in the cyanobacterium Synechocystis sp. PCC 6803, and analyzed isolated PSII core complexes using Fourier transform infrared (FTIR) difference spectroscopy and mass spectrometry (MS). The S2-minus-S1 FTIR difference spectrum of the PSII complexes of the D1-D170H mutant showed features virtually identical to those of the wild-type PSII. MS analysis further showed that ~70% of D1 proteins from the PSII complexes of D1-D170H possessed the wild-type amino acid sequence, although only the mutated sequence was detected in genomic DNA in the same batch of cells for PSII preparations. In contrast, a D1-S169A mutant as a control showed a modified FTIR spectrum and only a mutated D1 protein. It is thus concluded that the FTIR spectrum of the D1-D170H mutant actually reflects that of wild-type PSII, whereas the Mn4CaO5 cluster is not formed in PSII with D1-D170H mutation. Although the mechanism of production of the wild-type D1 protein in the D1-D170H mutant is unknown at present, a caution is necessary in the analysis of site-directed mutants of crucial residues in the D1 protein, and mutation has to be confirmed not only at the DNA level but also at the amino acid level.

Original languageEnglish
Article number148086
JournalBiochimica et Biophysica Acta - Bioenergetics
Volume1861
Issue number1
DOIs
Publication statusPublished - Jan 1 2020

Keywords

  • Carboxylate ligand
  • Fourier transform infrared spectroscopy
  • Mass spectrometry
  • Oxygen evolution
  • Photosystem II
  • Water oxidation

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Cell Biology

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