Biochemical and crystallographic characterization of ferredoxin-NADP(+) reductase from nonphotosynthetic tissues.
Distinct forms of ferredoxin-NADP(+) reductase are expressed in photosynthetic and nonphotosynthetic plant tissues. Both enzymes catalyze electron transfer between NADP(H) and ferredoxin; whereas in leaves the enzyme transfers reducing equivalents from photoreduced ferredoxin to NADP(+) in photosynthesis, in roots it has the opposite physiological role, reducing ferredoxin at the expense of NADPH mainly for use in nitrate assimilation. Here, structural and kinetic properties of a nonphotosynthetic isoform were analyzed to define characteristics that may be related to tissue-specific function. Compared with spinach leaf ferredoxin-NADP(+) reductase, the recombinant corn root isoform showed a slightly altered absorption spectrum, a higher pI, a >30-fold higher affinity for NADP(+), greater susceptibility to limited proteolysis, and an approximately 20 mV more positive redox potential. The 1.7 A resolution crystal structure is very similar to the structures of ferredoxin-NADP(+) reductases from photosynthetic tissues. Four distinct structural features of this root ferredoxin-NADP(+) reductases are an alternate conformation of the bound FAD molecule, an alternate path for the amino-terminal extension, a disulfide bond in the FAD-binding domain, and changes in the surface that binds ferredoxin.
Aliverti
A
A
Dipartimento di Fisiologia e Biochimica Generali, Università degli Studi di Milano, Via Celoria 26, 20133 Milano, Italy.
Faber
R
R
Finnerty
C M
CM
Ferioli
C
C
Pandini
V
V
Negri
A
A
Karplus
P A
PA
Zanetti
G
G
D018360:Crystallography, X-Ray; D017931:DNA Primers / Q000737:chemistry; D004586:Electrophoresis; D005287:Ferredoxin-NADP Reductase / Q000737:chemistry* / Q000502:physiology; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D005822:Genetic Vectors; D009249:NADP / Q000378:metabolism; D010084:Oxidation-Reduction; D018515:Plant Leaves / Q000201:enzymology; D018517:Plant Roots / Q000201:enzymology*; D011487:Protein Conformation; D020033:Protein Isoforms / Q000502:physiology; D013057:Spectrum Analysis; D018724:Spinacia oleracea / Q000201:enzymology; D013329:Structure-Activity Relationship; D003313:Zea mays / Q000201:enzymology*
D017931:DNA Primers; D020033:Protein Isoforms; D005182:Flavin-Adenine Dinucleotide; D009249:NADP; D005287:Ferredoxin-NADP Reductase