Characterization of site-directed mutants in manganese-stabilizing protein (MSP) of Synechocystis sp. PCC6803 unable to grow photoautotrophically in the absence of cytochrome c-550

Sufian F. Al-Khaldi, James Coker, Jian-Ren Shen, Robert L. Burnap

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8 Citations (Scopus)

Abstract

To investigate the interaction between the manganese-stabilizing protein (MSP) and cytochrome c-550 (cyt. c550) of the photosystem II (PSII) complex in the cyanobacterium Synechocystis sp. PCC6803, three site-directed amino acid substitution mutants in MSP (MSP-D159N, MSP-R163L, MSP-D159N/R163L) were created by single and double amino acid substitution mutagenesis. The modified psbO genes encoding the mutants forms of MSP were used to transform a single-deletion mutant ΔpsO strain lacking MSP as well as a double-deletion strain ΔpsbO:ΔpsbV lacking both MSP and cyt. c-550. The mutant forms of MSP were expressed in each case and all permitted autotrophic growth in strains expressing cyt. c-550. However, when the MSP mutations were introduced into a strain which lacks cyt. c-550 (ΔpsbV), the two single amino acid substitution mutants (ΔpsbV:MSP-D159N and ΔpsbV:MSP-R163L) failed to grow photoautotrophically. These strains exhibited coupled O2-evolving activity of 68-77% compared to the wild-type control using CO2 as an electron acceptor and maximal uncoupled O2-evolution rates of 42-57% using 2,6-dichloro-p-benzoquinone (DCBQ) as an artificial electron acceptor. Interestingly, when the two amino acid substitutions were together in the absence of cyt. c-550 (ΔApsbV:MSP-D 159N/R163L), the mutant grew photoautotrophically and the oxygen-evolving activities were higher than in the single mutants. This indicates that the MSP-DI59N mutant suppresses the non-autotrophic phenotype of MSP-R163L (or vice versa) in the absence of cyt. c-550. The possibilities of a direct (ionic) or indirect interaction between D159 and R163 of MSP are discussed.

Original languageEnglish
Pages (from-to)33-41
Number of pages9
JournalPlant Molecular Biology
Volume43
Issue number1
DOIs
Publication statusPublished - 2000
Externally publishedYes

Fingerprint

Synechocystis
cytochrome c
Manganese
Cytochromes c
manganese
mutants
Proteins
proteins
amino acid substitution
Amino Acid Substitution
Substitution reactions
Amino Acids
Autotrophic Processes
electrons
Electrons
benzoquinones
Mutagenesis
Gene encoding
Photosystem II Protein Complex
Cyanobacteria

Keywords

  • Cytochrome c-550
  • Manganese-stabilizing protein (MSP)
  • Photosynthesis
  • PSII
  • Synechocystis sp. PCC6803

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)
  • Biochemistry

Cite this

@article{b2868fed09814031a36b2e570a0654b0,
title = "Characterization of site-directed mutants in manganese-stabilizing protein (MSP) of Synechocystis sp. PCC6803 unable to grow photoautotrophically in the absence of cytochrome c-550",
abstract = "To investigate the interaction between the manganese-stabilizing protein (MSP) and cytochrome c-550 (cyt. c550) of the photosystem II (PSII) complex in the cyanobacterium Synechocystis sp. PCC6803, three site-directed amino acid substitution mutants in MSP (MSP-D159N, MSP-R163L, MSP-D159N/R163L) were created by single and double amino acid substitution mutagenesis. The modified psbO genes encoding the mutants forms of MSP were used to transform a single-deletion mutant ΔpsO strain lacking MSP as well as a double-deletion strain ΔpsbO:ΔpsbV lacking both MSP and cyt. c-550. The mutant forms of MSP were expressed in each case and all permitted autotrophic growth in strains expressing cyt. c-550. However, when the MSP mutations were introduced into a strain which lacks cyt. c-550 (ΔpsbV), the two single amino acid substitution mutants (ΔpsbV:MSP-D159N and ΔpsbV:MSP-R163L) failed to grow photoautotrophically. These strains exhibited coupled O2-evolving activity of 68-77{\%} compared to the wild-type control using CO2 as an electron acceptor and maximal uncoupled O2-evolution rates of 42-57{\%} using 2,6-dichloro-p-benzoquinone (DCBQ) as an artificial electron acceptor. Interestingly, when the two amino acid substitutions were together in the absence of cyt. c-550 (ΔApsbV:MSP-D 159N/R163L), the mutant grew photoautotrophically and the oxygen-evolving activities were higher than in the single mutants. This indicates that the MSP-DI59N mutant suppresses the non-autotrophic phenotype of MSP-R163L (or vice versa) in the absence of cyt. c-550. The possibilities of a direct (ionic) or indirect interaction between D159 and R163 of MSP are discussed.",
keywords = "Cytochrome c-550, Manganese-stabilizing protein (MSP), Photosynthesis, PSII, Synechocystis sp. PCC6803",
author = "Al-Khaldi, {Sufian F.} and James Coker and Jian-Ren Shen and Burnap, {Robert L.}",
year = "2000",
doi = "10.1023/A:1006419928712",
language = "English",
volume = "43",
pages = "33--41",
journal = "Plant Molecular Biology",
issn = "0167-4412",
publisher = "Springer Netherlands",
number = "1",

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TY - JOUR

T1 - Characterization of site-directed mutants in manganese-stabilizing protein (MSP) of Synechocystis sp. PCC6803 unable to grow photoautotrophically in the absence of cytochrome c-550

AU - Al-Khaldi, Sufian F.

AU - Coker, James

AU - Shen, Jian-Ren

AU - Burnap, Robert L.

PY - 2000

Y1 - 2000

N2 - To investigate the interaction between the manganese-stabilizing protein (MSP) and cytochrome c-550 (cyt. c550) of the photosystem II (PSII) complex in the cyanobacterium Synechocystis sp. PCC6803, three site-directed amino acid substitution mutants in MSP (MSP-D159N, MSP-R163L, MSP-D159N/R163L) were created by single and double amino acid substitution mutagenesis. The modified psbO genes encoding the mutants forms of MSP were used to transform a single-deletion mutant ΔpsO strain lacking MSP as well as a double-deletion strain ΔpsbO:ΔpsbV lacking both MSP and cyt. c-550. The mutant forms of MSP were expressed in each case and all permitted autotrophic growth in strains expressing cyt. c-550. However, when the MSP mutations were introduced into a strain which lacks cyt. c-550 (ΔpsbV), the two single amino acid substitution mutants (ΔpsbV:MSP-D159N and ΔpsbV:MSP-R163L) failed to grow photoautotrophically. These strains exhibited coupled O2-evolving activity of 68-77% compared to the wild-type control using CO2 as an electron acceptor and maximal uncoupled O2-evolution rates of 42-57% using 2,6-dichloro-p-benzoquinone (DCBQ) as an artificial electron acceptor. Interestingly, when the two amino acid substitutions were together in the absence of cyt. c-550 (ΔApsbV:MSP-D 159N/R163L), the mutant grew photoautotrophically and the oxygen-evolving activities were higher than in the single mutants. This indicates that the MSP-DI59N mutant suppresses the non-autotrophic phenotype of MSP-R163L (or vice versa) in the absence of cyt. c-550. The possibilities of a direct (ionic) or indirect interaction between D159 and R163 of MSP are discussed.

AB - To investigate the interaction between the manganese-stabilizing protein (MSP) and cytochrome c-550 (cyt. c550) of the photosystem II (PSII) complex in the cyanobacterium Synechocystis sp. PCC6803, three site-directed amino acid substitution mutants in MSP (MSP-D159N, MSP-R163L, MSP-D159N/R163L) were created by single and double amino acid substitution mutagenesis. The modified psbO genes encoding the mutants forms of MSP were used to transform a single-deletion mutant ΔpsO strain lacking MSP as well as a double-deletion strain ΔpsbO:ΔpsbV lacking both MSP and cyt. c-550. The mutant forms of MSP were expressed in each case and all permitted autotrophic growth in strains expressing cyt. c-550. However, when the MSP mutations were introduced into a strain which lacks cyt. c-550 (ΔpsbV), the two single amino acid substitution mutants (ΔpsbV:MSP-D159N and ΔpsbV:MSP-R163L) failed to grow photoautotrophically. These strains exhibited coupled O2-evolving activity of 68-77% compared to the wild-type control using CO2 as an electron acceptor and maximal uncoupled O2-evolution rates of 42-57% using 2,6-dichloro-p-benzoquinone (DCBQ) as an artificial electron acceptor. Interestingly, when the two amino acid substitutions were together in the absence of cyt. c-550 (ΔApsbV:MSP-D 159N/R163L), the mutant grew photoautotrophically and the oxygen-evolving activities were higher than in the single mutants. This indicates that the MSP-DI59N mutant suppresses the non-autotrophic phenotype of MSP-R163L (or vice versa) in the absence of cyt. c-550. The possibilities of a direct (ionic) or indirect interaction between D159 and R163 of MSP are discussed.

KW - Cytochrome c-550

KW - Manganese-stabilizing protein (MSP)

KW - Photosynthesis

KW - PSII

KW - Synechocystis sp. PCC6803

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U2 - 10.1023/A:1006419928712

DO - 10.1023/A:1006419928712

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