Occurrence of ferredoxin:NAD(+) oxidoreductase activity and its ion specificity in several Gram-positive and Gram-negative bacteria

Abstract

A ferredoxin:NAD(+) oxidoreductase was recently discovered as a redox-driven ion pump in the anaerobic, acetogenic bacterium Acetobacterium woodii. The enzyme is assumed to be encoded by the rnf genes. Since these genes are present in the genomes of many bacteria, we tested for ferredoxin:NAD(+) oxidoreductase activity in cytoplasmic membranes from several different Gram-positive and Gram-negative bacteria that have annotated rnf genes. We found this activity in Clostridium tetanomorphum, Clostridium ljungdahlii, Bacteroides fragilis, and Vibrio cholerae but not in Escherichia coli and Rhodobacter capsulatus. As in A. woodii, the activity was Na(+)-dependent in C. tetanomorphum and B. fragilis but Na(+)-independent in C. ljungdahlii and V. cholerae. We deleted the rnf genes from B. fragilis and demonstrated that the mutant has greatly reduced ferredoxin:NAD(+) oxidoreductase activity. This is the first genetic proof that the rnf genes indeed encode the reduced ferredoxin:NAD(+) oxidoreductase activity.

Keywords: Energy conservation; Fdred:NAD+ oxidoreductase; Ion pump; Rnf.

Figure 1. Arrangement of the rnf genes in different bacteria.

Figure 2. Fd_red:NAD⁺ oxidoreductase activity of membranes of C. ljungdahlii as a function of Na⁺ concentration (A) and of inverted membrane vesicles in the presence of different ionophores (B).

Fno activity was measured in anoxic cuvettes filled with 1 ml 20 mM Tris (sodium free)-HCl buffer (pH 7.7) containing 2 mM DTE and 2 μM resazurin at a pressure of 0.5 × 10⁵ Pa CO. NaCl was added to the concentration indicated. Ferredoxin (30 μM), Acs/CODH (30 μg/ml), and washed membranes or inverted membrane vesicles (150 μg/ml) were added. If indicated, the ionophores ETH2120 or TCS were added at a concentration of 10 μM. The reaction was started by addition of NAD⁺ (4 mM). Formation of NADH was measured at 340 nm.