Measurement of photosystem I activity with photoreduction of recombinant flavodoxin

Jindong Zhao, Ronggui Li, Donald Ashley Bryant

Research output: Contribution to journalArticle

11 Citations (Scopus)

Abstract

Flavodoxin can function as an alternative electron acceptor for photosystem I (PSI) in place of ferredoxin under iron-limiting conditions. The isiB gene, encoding the flavodoxin in Synechococcus sp. PCC 7002, was overexpressed in Escherichia coli. Under the conditions employed, most recombinant flavodoxin (rFlvd) was in soluble form with cofactor correctly inserted. The absorption spectrum of rFlvd was identical to that of the native flavodoxin of the cyanobacteria. Photoreduction of rFlvd by PSI particles and thylakoid membranes was determined directly by monitoring the absorption change at 467 nm. The optimal conditions for rFlvd photoreduction were determined. Compared to other methods currently employed to measure PSI activity such as oxygen uptake in the presence of methyl viologen and NADP+ photoreduction in the presence of ferredoxin and ferredoxin:NADP+ oxidoreductase, measurement of PSI activity with flavodoxin as an electron acceptor has several advantages. It measures the full-chain electron transfer chain of PSI since flavodoxin accepts electrons from F(A)/F(B) and it is much simpler than the method with NADP+ photoreduction. With this method, we found that the affinity of wild-type PSI for rFlvd was 35% higher than that of the PsaE-less PSI, showing that this method is sensitive to structural changes of PSI. Our results demonstrate that rFlvd photoreduction is an effective and simple method for PSI activity measurement.

Original languageEnglish (US)
Pages (from-to)263-270
Number of pages8
JournalAnalytical Biochemistry
Volume264
Issue number2
DOIs
StatePublished - Nov 15 1998

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Flavodoxin
Photosystem I Protein Complex
Electrons
Ferredoxins
NADP
Ferredoxin-NADP Reductase
Synechococcus
Thylakoids
Paraquat
Gene encoding
Cyanobacteria
Escherichia coli
Absorption spectra

All Science Journal Classification (ASJC) codes

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

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title = "Measurement of photosystem I activity with photoreduction of recombinant flavodoxin",
abstract = "Flavodoxin can function as an alternative electron acceptor for photosystem I (PSI) in place of ferredoxin under iron-limiting conditions. The isiB gene, encoding the flavodoxin in Synechococcus sp. PCC 7002, was overexpressed in Escherichia coli. Under the conditions employed, most recombinant flavodoxin (rFlvd) was in soluble form with cofactor correctly inserted. The absorption spectrum of rFlvd was identical to that of the native flavodoxin of the cyanobacteria. Photoreduction of rFlvd by PSI particles and thylakoid membranes was determined directly by monitoring the absorption change at 467 nm. The optimal conditions for rFlvd photoreduction were determined. Compared to other methods currently employed to measure PSI activity such as oxygen uptake in the presence of methyl viologen and NADP+ photoreduction in the presence of ferredoxin and ferredoxin:NADP+ oxidoreductase, measurement of PSI activity with flavodoxin as an electron acceptor has several advantages. It measures the full-chain electron transfer chain of PSI since flavodoxin accepts electrons from F(A)/F(B) and it is much simpler than the method with NADP+ photoreduction. With this method, we found that the affinity of wild-type PSI for rFlvd was 35{\%} higher than that of the PsaE-less PSI, showing that this method is sensitive to structural changes of PSI. Our results demonstrate that rFlvd photoreduction is an effective and simple method for PSI activity measurement.",
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Measurement of photosystem I activity with photoreduction of recombinant flavodoxin. / Zhao, Jindong; Li, Ronggui; Bryant, Donald Ashley.

In: Analytical Biochemistry, Vol. 264, No. 2, 15.11.1998, p. 263-270.

Research output: Contribution to journalArticle

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