Purification and characterization of ferredoxin-nicotinamide adenine dinucleotide phosphate reductase from a nitrogen-fixing bacterium

Area of Science:

• Biochemistry
• Enzymology
• Microbial Metabolism

Background:

• Bacillus polymyxa nitrogenase requires NADPH for reducing power.
• Ferredoxin-NADP(+) reductase (FNR) is essential for electron transfer in many organisms.
• Previous studies suggested the presence of FNR activity in B. polymyxa.

Purpose of the Study:

• To provide direct evidence for and characterize the ferredoxin-NADP(+) reductase in Bacillus polymyxa.
• To investigate the enzyme's purification, properties, and substrate specificity.

Main Methods:

• Enzyme purification using high-speed centrifugation, ammonium sulfate fractionation, and chromatography (Sephadex G-100, DEAE-cellulose).
• Molecular weight determination by gel filtration.
• Enzyme characterization including polyacrylamide gel electrophoresis, substrate specificity assays, and pH optimum determination.

Main Results:

• Ferredoxin-NADP(+) reductase was purified approximately 80-fold and identified as a flavoprotein with a molecular weight of 60,000.
• The enzyme showed specificity for NADPH as the electron donor and NADP(+) as the acceptor.
• It catalyzed electron transfer to various ferredoxins (B. polymyxa, Clostridium pasteurianum, Azotobacter vinelandii, spinach) and accepted electrons from photoreduced ferredoxin.
• The reductase also exhibited irreversible NADPH-NAD(+) transhydrogenase activity.

Conclusions:

• Bacillus polymyxa possesses an active ferredoxin-NADP(+) reductase essential for its nitrogenase system.
• The enzyme shares similarities with chloroplast ferredoxin-NADP(+) reductase in terms of pH optima and substrate interactions.
• The broad specificity suggests a significant role in the organism's redox metabolism.