Abstract
The ferredoxin-dependent nitrite reductase from the green alga Chlamydomonas reinhardtii has been cloned, expressed in Escherichia coli as a His-tagged recombinant protein, and purified to homogeneity. The spectra, kinetic properties and substrate-binding parameters of the C. reinhardtii enzyme are quite similar to those of the ferredoxin-dependent spinach chloroplast nitrite reductase. Computer modeling, based on the published structure of spinach nitrite reductase, predicts that the structure of C. reinhardtii nitrite reductase will be similar to that of the spinach enzyme. Chemical modification studies and the ionic-strength dependence of the enzyme's ability to interact with ferredoxin are consistent with the involvement of arginine and lysine residues on C. reinhardtii nitrite reductase in electrostatically-stabilized binding to ferredoxin. The C. reinhardtii enzyme has been used to demonstrate that hydroxylamine can serve as an electron-accepting substrate for the enzyme and that the product of hydroxylamine reduction is ammonia, providing the first experimental evidence for the hypothesis that hydroxylamine, bound to the enzyme, can serve as a late intermediate during the reduction of nitrite to ammonia catalyzed by the enzyme.
| Original language | English |
|---|---|
| Pages (from-to) | 67-77 |
| Number of pages | 11 |
| Journal | Photosynthesis Research |
| Volume | 103 |
| Issue number | 2 |
| DOIs | |
| Publication status | Published - Jan 2010 |
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Keywords
- Ferredoxin
- Hydroxylamine reduction
- NII1
- Nitrite reductase
- PETF
- Tertiary structure
ASJC Scopus subject areas
- Plant Science
- Cell Biology
- Biochemistry
Cite this
Enzymatic properties of the ferredoxin-dependent nitrite reductase from chlamydomonas reinhardtii. Evidence for hydroxylamine as a late intermediate in ammonia production. / Hirasawa, Masakazu; Tripathy, Jatindra N.; Sommer, Frederik; Somasundaram, Ramasamy; Chung, Jung Sung; Nestander, Matthew; Kruthiventi, Mahima; Zabet-Moghaddam, Masoud; Johnson, Michael K.; Merchant, Sabeeha S.; Allen, James Paul; Knaff, David B.
In: Photosynthesis Research, Vol. 103, No. 2, 01.2010, p. 67-77.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Enzymatic properties of the ferredoxin-dependent nitrite reductase from chlamydomonas reinhardtii. Evidence for hydroxylamine as a late intermediate in ammonia production
AU - Hirasawa, Masakazu
AU - Tripathy, Jatindra N.
AU - Sommer, Frederik
AU - Somasundaram, Ramasamy
AU - Chung, Jung Sung
AU - Nestander, Matthew
AU - Kruthiventi, Mahima
AU - Zabet-Moghaddam, Masoud
AU - Johnson, Michael K.
AU - Merchant, Sabeeha S.
AU - Allen, James Paul
AU - Knaff, David B.
PY - 2010/1
Y1 - 2010/1
N2 - The ferredoxin-dependent nitrite reductase from the green alga Chlamydomonas reinhardtii has been cloned, expressed in Escherichia coli as a His-tagged recombinant protein, and purified to homogeneity. The spectra, kinetic properties and substrate-binding parameters of the C. reinhardtii enzyme are quite similar to those of the ferredoxin-dependent spinach chloroplast nitrite reductase. Computer modeling, based on the published structure of spinach nitrite reductase, predicts that the structure of C. reinhardtii nitrite reductase will be similar to that of the spinach enzyme. Chemical modification studies and the ionic-strength dependence of the enzyme's ability to interact with ferredoxin are consistent with the involvement of arginine and lysine residues on C. reinhardtii nitrite reductase in electrostatically-stabilized binding to ferredoxin. The C. reinhardtii enzyme has been used to demonstrate that hydroxylamine can serve as an electron-accepting substrate for the enzyme and that the product of hydroxylamine reduction is ammonia, providing the first experimental evidence for the hypothesis that hydroxylamine, bound to the enzyme, can serve as a late intermediate during the reduction of nitrite to ammonia catalyzed by the enzyme.
AB - The ferredoxin-dependent nitrite reductase from the green alga Chlamydomonas reinhardtii has been cloned, expressed in Escherichia coli as a His-tagged recombinant protein, and purified to homogeneity. The spectra, kinetic properties and substrate-binding parameters of the C. reinhardtii enzyme are quite similar to those of the ferredoxin-dependent spinach chloroplast nitrite reductase. Computer modeling, based on the published structure of spinach nitrite reductase, predicts that the structure of C. reinhardtii nitrite reductase will be similar to that of the spinach enzyme. Chemical modification studies and the ionic-strength dependence of the enzyme's ability to interact with ferredoxin are consistent with the involvement of arginine and lysine residues on C. reinhardtii nitrite reductase in electrostatically-stabilized binding to ferredoxin. The C. reinhardtii enzyme has been used to demonstrate that hydroxylamine can serve as an electron-accepting substrate for the enzyme and that the product of hydroxylamine reduction is ammonia, providing the first experimental evidence for the hypothesis that hydroxylamine, bound to the enzyme, can serve as a late intermediate during the reduction of nitrite to ammonia catalyzed by the enzyme.
KW - Ferredoxin
KW - Hydroxylamine reduction
KW - NII1
KW - Nitrite reductase
KW - PETF
KW - Tertiary structure
UR - http://www.scopus.com/inward/record.url?scp=76149098783&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=76149098783&partnerID=8YFLogxK
U2 - 10.1007/s11120-009-9512-5
DO - 10.1007/s11120-009-9512-5
M3 - Article
VL - 103
SP - 67
EP - 77
JO - Photosynthesis Research
JF - Photosynthesis Research
SN - 0166-8595
IS - 2
ER -