Ferredoxin-NADP+ reductase from Pseudomonas putida functions as a ferric reductase

Area of Science:

• Microbiology
• Biochemistry
• Enzymology

Background:

• Pseudomonas putida possesses two uncharacterized ferredoxin-NADP(+) reductases (Fprs).
• Ferric reductase activity is structurally related to the Fpr superfamily, but not explicitly annotated in P. putida.
• Understanding Fpr functions is crucial for elucidating iron metabolism and redox processes in P. putida.

Purpose of the Study:

• To investigate the biochemical and physiological functions of FprA and FprB in Pseudomonas putida.
• To determine the roles of FprA and FprB as ferric and flavin reductases.
• To assess the contribution of Fprs to bacterial growth under iron stress.

Main Methods:

• Biochemical assays (ferric reductase, flavin reductase) using wild-type and mutant P. putida strains.
• Enzyme kinetics analysis (kcat/Km) with native and synthetic ferric chelators and flavin mononucleotide (FMN).
• Growth rate measurements in iron-containing minimal media and homology modeling of enzyme structures.

Main Results:

• FprA and FprB exhibit distinct substrate preferences, utilizing NADPH and NADH respectively.
• Both Fprs prefer native ferric chelators and use free FMN as an electron carrier, with FprB showing higher efficiency.
• fprB mutants display significantly reduced growth rates in iron-limited conditions, indicating a critical role for FprB.

Conclusions:

• This study establishes the flavin and ferric reductase activities of FprA and FprB in P. putida.
• FprB is identified as a key NADH-dependent ferric/flavin reductase, particularly important under iron stress.
• The findings suggest potential redundancy or alternative pathways for NADPH-dependent flavin reduction.