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Iron-sulfur clusters ([Fe-The in vivo maturation of simple [Fe-S] proteins is proposed to require preassembly of [Fe-S] species on molecular scaffolds. The first [Fe-S] cluster assembly system to be described is the NIF 2 system from Azotobacter vinelandii. This system consists of a cysteine desulfurase, encoded by nifS, which supplies the S for [Fe-S] cluster formation, and a proposed scaffold protein, encoded by nifU (1). The NIF system is specialized for the maturation of [Fe-S] proteins involved in nitrogen fixation.A. vinelandii also contains a second [Fe-S] protein maturation system designated ISC. The ISC system is required for the general maturation of cellular [Fe-S] proteins involved in intermediary metabolism, such as aconitase (2). The ISC system is more complicated than the NIF system as it includes the products of eight contiguous genes : iscR, iscS, iscU, iscA, hscB, hscA, fdx, and iscX (3). Although the NIF and ISC systems exhibit physiological target specificity, each can partially replace the function of the other, when expressed at high levels (4, 5).Even though the NIF and ISC systems are differentiated by their apparent target specificities, they share a number of common structural and functional features. For example, NifS and IscS have similar sequences, and they both exhibit cysteine desulfurase activity (2). IscU also shares considerable sequence identity when compared with the N-terminal domain of NifU, including conservation of three cysteine residues that are likely to provide the nucleation site(s) for [Fe-S] cluster assembly (2, 6).NifU is a modular protein that contains three distinct domains (Fig. 1). The central domain contains a stable redoxactive [2Fe-2S] cluster with an as-yet-unknown function (7). In vitro and in vivo experiments have established that labile [Fe-S] clusters can be assembled on both the N-terminal and C-terminal domains of NifU, and such cluster-loaded forms of NifU can be used for activation of the nitrogenase 9). Thus, NifU contains two different sites upon which labile [Fe-S] clusters can be assembled in vitro, but the functional relationship between these sites is not yet known.There are no genes within the ISC transcriptional unit that encode proteins with sequence similarity to the C-terminal domain of NifU. However, located elsewhere on the A. vinelandii genome is a gene, designated nfuA, whose product encodes a protein having a C-terminal sequence similar to the C-terminal domain of NifU (Fig. 1). The sequence conservation between NifU and NfuA includes two cysteine residues that are required for the in vitro assembly of [Fe-S] clusters within the NifU C-terminal domain. Like NifU, NfuA also appears to be a modular protein because the amino acid sequence within its N-terminal region shares some sequence similarity with another protein involved in [Fe-S] protein maturation designated IscA (Fig. 1). IscA is a nonessential protein encoded within the ISC transcriptional unit, and it has been proposed to serve as an alternative [Fe-S] cluster ass...

The H-cluster is a complex bridged metal assembly at the active site of [FeFe]-hydrogenases that consists of a [4Fe-4S] subcluster bridged to a 2Fe-containing subcluster with unique nonprotein ligands, including carbon monoxide, cyanide, and a dithiolate ligand of unknown composition. Specific biosynthetic gene products (HydE, HydF, and HydG) responsible for the biosynthesis of the H-cluster and the maturation of active [FeFe]-hydrogenase have previously been identified and shown to be required for the heterologous expression of active [FeFe]-hydrogenase [Posewitz, M. C., et al. (2004) J. Biol. Chem. 279, 25711-25720]. The precise roles of the maturation proteins are unknown; the most likely possibility is that they are directed at the synthesis of the entire 6Fe-containing H-cluster, the 2Fe subcluster, or only the unique ligands of the 2Fe subcluster. The spectroscopic and biochemical characterization of HydA(DeltaEFG) (the [FeFe]-hydrogenase structural protein expressed in the absence of the maturation machinery) reported here indicates that a [4Fe-4S] cluster is incorporated into the H-cluster site. The purified protein in a representative preparation contains Fe (3.1 +/- 0.5 Fe atoms per HydA(DeltaEFG)) and S(2-) (1.8 +/- 0.5 S(2-) atoms per HydA(DeltaEFG)) and exhibits UV-visible spectroscopic features characteristic of iron-sulfur clusters, including a bleaching of the visible chromophore upon addition of dithionite. The reduced protein gave rise to an axial S = (1)/(2) EPR signal (g = 2.04 and 1.91) characteristic of a reduced [4Fe-4S](+) cluster. Mossbauer spectroscopic characterization of (57)Fe-enriched HydA(DeltaEFG) provided further evidence of the presence of a redox active [4Fe-4S](2+/+) cluster. Iron K-edge EXAFS data provided yet further support for the presence of a [4Fe-4S] cluster in HydA(DeltaEFG). These spectroscopic studies were combined with in vitro activation studies that demonstrate that HydA(DeltaEFG) can be activated by the specific maturases only when a [4Fe-4S] cluster is present in the protein. In sum, this work supports a model in which the role of the maturation machinery is to synthesize and insert the 2Fe subcluster and/or its ligands and not the entire 6Fe-containing H-cluster bridged assembly.

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Mossbauer and electron paramagnetic resonance (EPR) data have recently been presented as evidence for a different type of metal cluster in a ferredoxin from Azotobacter vmnelandii (1). The same structure has also been reported for a ferredoxin (Fd II) from Desulfovibrio gigas (2) and for aconitase from beef heart mitochondria (3). The structure contains three spincoupled iron atoms (1, 2). X-ray crystallographic evidence (4) and chemical data (3) suggest that the center contains iron and sulfide in equimolar amounts; thus, a [3Fe-3S] cluster is anticipated.The function of the Azotobacter ferredoxin has not yet been established. The D. gigas ferredoxin appears to mediate electron transfer between cytochrome C3 and the sulfite reductase system (5), whereas a regulatory function has been proposed for the aconitase cluster (6). In the three proteins the new cluster can be prepared in two stable oxidation states. In the oxidized form the centers yield a EPR signal around g = 2.01 suggesting a system (cluster) spin S = 1/2. Upon a one-electron reduction the cluster becomes EPR-silent. According to the Mbssbauer data (1, 2), the reduced cluster has electronic spin S > 1. High-field Mossbauer studies have shown conclusively the presence of spin-exchange interactions among the iron atoms; spin coupling is evident in both oxidation states. Mbssbauer studies on the oxidized centers gave the following intriguing results: In the limit of fast electronic spin relaxation (at temperatures above 40 K) one sharp quadrupole doublet is observed, suggesting three indistinguishable iron sites. The observed isomer shift (6 = +0.27 mm/s relative to iron metal) and quadrupole splitting (AEQ = 0.63 mm/s for the Azotobacter ferredoxin and AEQ = 0.54 mm/s for Fd II) are typical of a high-spin ferric ion (S = 5/2) in a distorted tetrahedral environment of sulfur atoms. The prototype for such a coordination is rubredoxin (7). In the limit of slow electronic relaxation (T in which Si = 5/2 and i designates the iron sites. To a very good approximation the electron Zeeman interaction (gi = 2.0) and the magnetic hyperfine interaction are isotropic for high-spin ferric ions. We can ignore zero-field splitting terms in Eq. 1 because we will consider only ground multiplets with S = 1/2. In order to describe the exchange coupling among the three iron atoms we assume an interaction Hc = J12SI-S2 + J2SS2-Ss + Jl3Sl-SS.[2] The exchange interactions are assumed to be isotropic. Fur-thermore, terms quadratic in the Si are not considered. In order to account for the large differences of the observed magnetic hyperfine interactions (without invoking unreasonable covalencies) three distinct coupling constants are required.The data obtained for the two ferredoxins (1, 2) show that Hc is the dominant term in Eq. 1 and, therefore, we will consider Eq. 2 first. Because only isotropic exchange is considered, Ic can mix only states with the same total spin-i.e., S is a good quantum number. For Si = S2 = S3= 5/2 only two configurations with S = 1/2 occur i...