Mössbauer and EPR Evidence for an All-Ferrous Fe₄S₄ Cluster with S = 4 in the Fe Protein of Nitrogenase

Abstract

The ability to fix dinitrogen is restricted to a small but diverse group of procaryotes that contain the nitrogenase system. This system is composed of two proteins called the Fe protein and the MoFe protein; the latter contains the site of N 2 binding and reduction. 1,2 The Fe protein contains a single Fe 4 S 4 cluster bridged between two identical subunits each of which has a single binding site for MgATP. It is the only known reductant capable of reducing the MoFe protein such that the latter can reduce substrates. Our current understanding of nitrogenase is based on in vitro experiments with purified nitrogenase proteins using dithionite as the electron donor. Under these conditions the Fe 4 S 4 cluster shuttles between the 2+ and 1+ oxidation states.In 1994, Watt and Reddy 3 reported that the [Fe 4 S 4 ] 1+ Fe protein could be reversibly reduced (E°′ ) -460 mV vs SHE) to the all-ferrous [Fe 4 S 4 ] 0 state. This reaction was reported to be pH independent between pH 7.0 and 8.0. [Fe 4 S 4 ] 0 could be produced using methylviologen, Ti(III)citrate, or the physiological electron donor flavodoxin as reductants but could not be generated when dithionite was used as a reductant. 1,3,4 The proposal for the formation of an [Fe 4 S 4 ] 0 state was based on two main lines of evidence. 3 First, Coulometric reduction of the [Fe 4 S 4 ] 1+ state of the Fe protein showed that a second electron could be added to the protein but did not show whether the reduction was metal-centered. 5 Second, the two-electron reduced Fe protein did not exhibit the S ) 1 / 2 EPR signal that arises from the [Fe 4 S 4 ] 1+ state, but that signal could be elicited by adding 1 equiv of oxidant to the fully reduced protein. These observations suggest, but do not prove, the unprecedented formation 6 of a protein-bound all-ferrous Fe 4 S 4 cluster.For the Mössbauer and EPR studies we produced the twoelectron reduced form of the Fe protein by treating 57 Fe-enriched Azotobacter Vinelandii Fe protein (AV2) with Ti(III) citrate. 8 Figure 1 shows two Mössbauer spectra of Ti(III) citrate-reduced AV2. The zero-field spectra exhibit quadrupole doublets down to 4.2 K. Preliminary analysis of the whole data set (30 spectra) revealed four distinct sites, all with isomeric shift δ ) 0.68 mm/s. The simulation of Figure 1A assumes four doublets of equal intensity with ∆E Q ) 1.25, 1.40, 1.75, and 3.08 mm/s. The absence of magnetic features in the 4.2 K zero-field spectrum strongly suggests that the Fe 4 S 4 cluster has integer or zero spin. The isomer shift, δ, is an excellent indicator for the oxidation state of an Fe 4 S 4 cluster; typically, the average value of δ increases by 0.10-0.12 mm/s per electron added. Table 1 shows that the isomeric shifts of all iron sites are larger than those of the ferrous pair (δ ) 0.59 mm/s 12 ) of the S ) 1 / 2 state of [Fe 4 S 4 ] 1+ AV2. From these observations we conclude that all iron sites of the cluster are ferrous.As shown in Figure 1B, a weak applied magnetic field elicits substantial 57 Fe magnetic hyperfine inter...

Formation and characterization of an all-ferrous Rieske cluster and stabilization of the [2Fe-2S] 0 core by protonation

“…Its spectrum is similar to the spectra of the [2Fe-2S] 0 state of spinach ferredoxin, generated by complexation to a chromium reductant (12), and the [3Fe-4S] 2Ϫ -3H ϩ cluster in Sulfolobus acidocaldarius ferredoxin (3). Interestingly, the all-ferrous Fe-protein is distinct because it is pink-red, due to an absorption band at Ϸ520 nm (19,22). The all-ferrous cluster of BtRp is EPR silent, in both perpendicular and parallel mode, indicating that it has zero, or integer, spin.…”

The FeMoco-deficient MoFe Protein Produced by a nifHDeletion Strain of Azotobacter vinelandii Shows Unusual P-cluster Features

“…The (42,62). This finding raised the question as to whether it is also possible to reduce the metal centers of the ⌬nifH MoFe and ⌬nifB MoFe protein beyond the dithionitereduced state as was isolated.…”

“…Subsequently, indigo disulfonate was removed by a single passage over an anion-exchange column as described elsewhere (41). Ti(III) citrate and the Ti(III) citrate-reduced samples were prepared according to published methods (42,43). Ti(III) citrate, like Na 2 S 2 O 4 , was removed on a Sephadex G-25 column.…”

Flavodoxin hydroquinone reduces Azotobacter vinelandii Fe protein to the all-ferrous redox state with a S = 0 spin state

“…The discovery (4) and characterization (5)(6)(7)(8)(9)(10)(11)(12) of an [Fe 4 S 4 ] 0 Av2 has raised questions regarding how nitrogenase functions (13)(14)(15) because the 2ϩ͞0 couple involves two electrons instead of one (5)(6)(7) to decrease the optimal ATP requirement 2-fold. This energy efficiency makes the study of [Fe 4 (17), and an E m ϭ -790 mV (18) was estimated for the 1ϩ͞0 couple, which is in line with E m estimates of all-ferrous Fe-S clusters by using discrete Fourier transform calculations (19).…”