Kinetic analysis of the oxidative conversion of the [4Fe-4S]2+ cluster of FNR to a [2Fe-2S]2+ Cluster

Abstract

The ability of FNR to sense and respond to cellular O(2) levels depends on its [4Fe-4S](2+) cluster. In the presence of O(2), the [4Fe-4S](2+) cluster is converted to a [2Fe-2S](2+) cluster, which inactivates FNR as a transcriptional regulator. In this study, we demonstrate that approximately 2 Fe(2+) ions are released from the reaction of O(2) with the [4Fe-4S](2+) cluster. Fe(2+) release was then used as an assay of reaction progress to investigate the rate of [4Fe-4S](2+) to [2Fe-2S](2+) cluster conversion in vitro. We also found that there was no detectable difference in the rate of O(2)-induced cluster conversion for FNR free in solution compared to its DNA-bound form. In addition, the rate of FNR inactivation was monitored in vivo by measuring the rate at which transcriptional regulation by FNR is lost upon the exposure of cells to O(2); a comparison of the in vitro and in vivo rates of conversion suggests that O(2)-induced cluster conversion is sufficient to explain FNR inactivation in cells. FNR protein levels were also compared for cells grown under aerobic and anaerobic conditions.

FIG. 1.

Release of Fe²⁺ from the [4Fe-4S]²⁺ cluster of FNR upon reaction with O₂. 4Fe-FNR (2 μM) was mixed with 160 (open square), 240 (filled circle), 320 (open diamond), or 400 μM (filled triangle) O₂ in the presence of 100 μM ferene at 25°C. Absorption at 593 nm indicates formation of the Fe²⁺-ferene complex. The single exponential curve fit for each condition is represented as a line; all fits produced an R² of ≥0.997.

FIG. 2.

Comparison of the rates of [4Fe-4S]²⁺ cluster conversion with the release of Fe²⁺. 4Fe-FNR (5 μM) was incubated in anaerobic buffer in the presence (filled circles) or absence (filled triangles) of 100 μM ferene at 25°C. At time zero, O₂ was introduced and the loss of the [4Fe-4S]²⁺ cluster was monitored as a decrease in absorption at 420 nm, and the release of Fe²⁺ was monitored as an increase in absorption at 593 nm.

FIG. 3.

Plot of k_obs versus [O₂] for the release of Fe²⁺ from the [4Fe-4S]²⁺ cluster of FNR upon reaction with O₂. The values of k₂ and K_d were calculated for the reaction of O₂ with 4Fe-FNR from the plot of k_obs versus [O₂] in Fig. 1.

FIG. 4.

The effect of DNA on the release of Fe²⁺ from 4Fe-FNR. 4Fe-FNR (2 μM) was incubated at 25°C in the presence (filled diamonds) or absence (open diamonds) of 3 μM dsDNA to permit the formation of FNR · DNA complexes and mixed with anaerobic ferene (100 μM) prior to the introduction of air.

FIG. 5.

Effect of O₂ on expression of P_ndh in cells grown at 37°C. Normalized β-galactosidase activity per milliliter of culture (A) and OD₆₀₀ (B) from PK3286 (fnr⁺, P_ndh-lacZ) grown under continuous anaerobic growth conditions (open diamonds) or exposed to O₂ at time zero (filled diamonds) at 37°C. A normalized β-galactosidase value of 1 represents 7 U/ml for anaerobic cells and 2.8 U/ml for O₂-exposed cells. The error bars represent the standard errors of triplicate samples.

FIG. 6.

Effect of O₂ on expression of P_ndh-lacZ in cells grown at 25°C. β-galactosidase activity per milliliter of culture (filled triangles) and OD₆₀₀ (open triangles) from PK3286 (fnr⁺, P_ndh-lacZ) grown under anaerobic growth conditions and then exposed to O₂ at time zero (arrow) at 25°C. The error bars represent the standard errors of triplicate samples.

FIG. 7.

Effect of O₂ on expression from P_dmsA-lacZ in cells grown at 37°C. β-Galactosidase activity per milliliter of culture was calculated before and after exposure to O₂ (at time zero; arrow) from strains PK3293, Δfnr (open triangles), and PK3292 (fnr⁺; filled triangles) grown at 37°C. The error bars represent the standard errors of triplicate samples.