Enzymatic properties of the ferredoxin-dependent nitrite reductase from Chlamydomonas reinhardtii. Evidence for hydroxylamine as a late intermediate in ammonia production.
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.
Hirasawa
Masakazu
M
Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, 79409-1061, USA.
Tripathy
Jatindra N
JN
Sommer
Frederik
F
Somasundaram
Ramasamy
R
Chung
Jung-Sung
JS
Nestander
Matthew
M
Kruthiventi
Mahima
M
Zabet-Moghaddam
Masoud
M
Johnson
Michael K
MK
Merchant
Sabeeha S
SS
Allen
James P
JP
Knaff
David B
DB
D000641:Ammonia / Q000378:metabolism*; D055162:Biocatalysis; D016825:Chlamydomonas reinhardtii / Q000201:enzymology*; D004578:Electron Spin Resonance Spectroscopy; D050900:Ferredoxin-Nitrite Reductase / Q000737:chemistry / Q000378:metabolism*; D005288:Ferredoxins / Q000378:metabolism; D019811:Hydroxylamine / Q000378:metabolism*; D008958:Models, Molecular; D009573:Nitrites / Q000378:metabolism; D009994:Osmolar Concentration; D010084:Oxidation-Reduction; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000378:metabolism; D018724:Spinacia oleracea / Q000201:enzymology
D005288:Ferredoxins; D009573:Nitrites; D011994:Recombinant Proteins; D019811:Hydroxylamine; D000641:Ammonia; D050900:Ferredoxin-Nitrite Reductase