Plant adenosine 5'-phosphosulfate reductase is a novel iron-sulfur protein.
Adenosine 5'-phosphosulfate reductase (APR) catalyzes the two-electron reduction of adenosine 5'-phosphosulfate to sulfite and AMP, which represents the key step of sulfate assimilation in higher plants. Recombinant APRs from both Lemna minor and Arabidopsis thaliana were overexpressed in Escherichia coli and isolated as yellow-brown proteins. UV-visible spectra of these recombinant proteins indicated the presence of iron-sulfur centers, whereas flavin was absent. This result was confirmed by quantitative analysis of iron and acid-labile sulfide, suggesting a [4Fe-4S] cluster as the cofactor. EPR spectroscopy of freshly purified enzyme showed, however, only a minor signal at g = 2.01. Therefore, Mössbauer spectra of (57)Fe-enriched APR were obtained at 4.2 K in magnetic fields of up to 7 tesla, which were assigned to a diamagnetic [4Fe-4S](2+) cluster. This cluster was unusual because only three of the iron sites exhibited the same Mössbauer parameters. The fourth iron site gave, because of the bistability of the fit, a significantly smaller isomer shift or larger quadrupole splitting than the other three sites. Thus, plant assimilatory APR represents a novel type of adenosine 5'-phosphosulfate reductase with a [4Fe-4S] center as the sole cofactor, which is clearly different from the dissimilatory adenosine 5'-phosphosulfate reductases found in sulfate reducing bacteria.
Kopriva
S
S
Institute of Plant Sciences, University of Bern, CH-3013 Bern, Switzerland. kopriva@uni-freiburg.de
Büchert
T
T
Fritz
G
G
Suter
M
M
Weber
M
M
Benda
R
R
Schaller
J
J
Feller
U
U
Schürmann
P
P
Schünemann
V
V
Trautwein
A X
AX
Kroneck
P M
PM
Brunold
C
C
D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000201:enzymology; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli / Q000378:metabolism; D007501:Iron / Q000378:metabolism; D007506:Iron-Sulfur Proteins / Q000737:chemistry* / Q000502:physiology; D007700:Kinetics; D019684:Magnoliopsida / Q000201:enzymology*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010088:Oxidoreductases / Q000737:chemistry* / Q000502:physiology*; D050862:Oxidoreductases Acting on Sulfur Group Donors; D010940:Plant Proteins / Q000378:metabolism; D010944:Plants / Q000201:enzymology*; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism; D013053:Spectrophotometry; D015204:Spectroscopy, Mossbauer; D013455:Sulfur / Q000378:metabolism; D013997:Time Factors; D014466:Ultraviolet Rays
D007506:Iron-Sulfur Proteins; D010940:Plant Proteins; D011994:Recombinant Proteins; D013455:Sulfur; D007501:Iron; D010088:Oxidoreductases; D050862:Oxidoreductases Acting on Sulfur Group Donors; C019618:adenylylsulfate reductase