In recent years, it has become evident that [Fe-S] proteins, such as hydrogenase, nitrogenase and aconitase, require a complex machinery to assemble and insert their associated [Fe-S] clusters. So far, three different types of [Fe-S] cluster biosynthetic systems have been identified and these have been designated nif, isc and suf. In the present work, we show that the nif-specific [Fe-S] cluster biosynthetic system from Azotobacter vinelandii, which is required for nitrogenase maturation, cannot functionally replace the isc [Fe-S] cluster system used for the maturation of other [Fe-S] proteins, such as aconitase. The results indicate that, in certain cases, [Fe-S] cluster biosynthetic machineries have evolved to perform only specialized functions.
- isc genes
Simple complexes of iron and inorganic sulphide ([Fe-S] clusters) are contained in a diverse group of proteins, called [Fe-S] proteins, which participate in a wide variety of cellular processes, including electron transfer, catalysis and regulation of gene expression. Such functional versatility of [Fe-S] proteins is related to the structural and electronic plasticity of their cognate [Fe-S] clusters. The most familiar [Fe-S] clusters include [2Fe-2S] and [4Fe-4S] clusters, which are usually covalently attached to their protein partners through cysteine mercaptide ligands. In spite of their structural simplicity, the formation and insertion of [Fe-S] clusters into their protein partners is a complicated process.
Initial insights about the pathway for [Fe-S] cluster assembly were gained through analysis of Azotobacter vinelandii genes required for activation of nitrogenase, the catalytic component of biological nitrogen fixation. Nitrogenase comprises two catalytic partners, called the Fe protein and the MoFe protein, and both of these are [Fe-S] proteins . A biochemical-genetic analysis of nitrogen-fixation-specific (nif) genes required for nitrogenase maturation revealed that two of them, nifU and nifS, are uniquely required for the activation of both the Fe and the MoFe proteins [2,3]. Subsequent studies suggested that NifS is a cysteine desulphurase that uses pyridoxal-phosphate chemistry to activate S in the form of an enzyme-bound persulphide  and that NifU provides a molecular scaffold for assembly of ‘transient’ [Fe-S] cluster units destined for nitrogenase maturation . Key observations used to validate this model include: (i) NifU and NifS are capable of forming a transient macromolecular complex , (ii) co-incubation of NifU and NifS in the presence of L-cysteine and Fe++ results in the formation of labile [Fe-S] clusters on NifU , (iii) [Fe-S] cluster-loaded NifU can be used for the effective in vitro activation of apo-Fe protein , and (iv) placement of certain amino acid substitutions within NifU results in trapping of the transient [Fe-S] cluster on the NifU scaffold , thereby compromising the capacity for both in vivo and in vitro nitrogenase activation .
The isc and suf systems also have [Fe-S] cluster biosynthetic functions
Although genetic inactivation of either NifU or NifS results in a significantly lowered capacity for the in vivo maturation of nitrogenase, loss of NifU or NifS function does not completely eliminate the capacity for nitrogen fixation . This result indicated that NifU or NifS activities could be replaced at low levels by some other cellular activities. A search for other cellular components having NifU-like and NifS-like activities resulted in the identification of a group of genes proposed to be required for the maturation of other [Fe-S] proteins, for example, aconitase, that are not related to nitrogen fixation. This gene cluster (Figure 1), designated ‘isc’ (iron-sulphur-cluster), encodes proteins having functions analogous to NifU (IscU) and NifS (IscS), as well as several other proteins including, an alternative scaffold (IscA), molecular chaperones (HscB and HscA), a ferredoxin (Fdx) and a negative regulator (IscR) . Genes encoding homologues to IscS, IscU, IscA, HscB, HscA and Fdx are widely distributed in nature and a variety of genetic studies have clearly implicated all of them in some aspect of the maturation of [Fe-S] proteins [9,10]. A third type of [Fe-S] protein maturation machinery was identified in Escherichia coli, which has been designated ‘suf’ . For E. coli, which also has an intact isc gene cluster, genetic and physiological studies have established that the isc system operates under ‘normal’ growth conditions, whereas the suf system operates under conditions of Fe limitation or oxygen stress . Although there appear to be specialized components that differentiate the three identified [Fe-S] cluster biosynthetic machineries, they are unified by an apparent requirement for a cysteine desulphurase and [Fe-S] cluster assembly scaffold.
The picture that has emerged concerning [Fe-S] protein maturation is that some organisms have generalized ‘housekeeping’ [Fe-S] cluster biosynthetic machinery, as well as other more ‘specialized’ [Fe-S] cluster biosynthetic machinery. However, whether or not a particular type of [Fe-S] cluster biosynthetic machinery, isc, suf or nif, operates in a ‘housekeeping’ capacity or in a ‘specialized’ capacity appears to depend on a particular organism. Indeed, for Helicobacter pylori and Thermatoga maritima the nif-like and suf-like systems, respectively, appear to be the only intact [Fe-S] cluster biosynthetic systems available to these organisms . Interesting questions attached to the role of various [Fe-S] cluster biosynthetic machineries involves their evolutionary and functional relationships.
Controlled expression of isc and nif genes
As mentioned above, the capacity for nifU or nifS deletion strains to fix nitrogen at a very low level indicated that their functions could be partially supplemented by some other [Fe-S] cluster biosynthetic systems . Although this hypothesis led to the discovery of the isc-gene cluster, the possibility that isc gene products can participate in the maturation of nitrogenase could not be directly tested because genetic experiments indicated that inactivation of the isc genes is lethal . This situation also precluded the opportunity to examine whether or not a nif-specific [Fe-S] cluster biosynthetic component can functionally replace an isc-specific component. To overcome this problem and to develop an opportunity to examine the specific functions of different components of the isc-specific [Fe-S] cluster biosynthetic machinery, we developed a method for the controlled expression of individual isc- and nif-specific components uncoupled from their normal regulatory components.
In A. vinelandii and many other bacteria, the isc genes are controlled by a negative feedback mechanism where the holo-form of an [Fe-S] cluster-containing regulatory protein (encoded by iscR, see Figure 1) represses transcription of the isc gene cluster [14,15]. The nif genes are controlled by a complex regulatory cascade involving regulatory proteins encoded by nifA and nifL and are only expressed under conditions that required nitrogen fixation . To uncouple isc- or nif-regulated components from their normal regulatory elements, the isolated genes were placed under the control of a sucrose-inducible (scr) promoter in vitro by using recombinant techniques and subsequently reintegrated into the chromosome in single copy by using reciprocal recombination (Figure 1). Details of the genetic constructions will be reported elsewhere. The scr promoter is negatively regulated by the availability of sucrose in the same way that the lac promoter is controlled by the availability of lactose. These constructions resulted in duplication of the genes of choice (Figure 1, strains DJ1421 and DJ1496), where expression of one copy is controlled by the normal regulatory elements and the scr promoter controls expression of the second copy.
NifU and NifS cannot functionally replace IscU and IscS
Control experiments established that genes regulated by scr are expressed at a relatively high level when sucrose is present in the growth medium but are not expressed at detectable levels in the absence of sucrose. This situation permitted the placement of deletions within the normal copy of a particular gene (see Figure 1, strains DJ1445 and DJ1475) whose function is replaced by the second copy, if cells are grown in the presence of sucrose under conditions where the function of the deleted gene is required. When sucrose is removed from the growth medium, in this case replaced by glucose, products of genes controlled by the scr promoter are gradually depleted from the cell. In this way, the physiological and biochemical consequences of the loss of function of a particular gene product can be unambiguously evaluated. The results of controlled expression experiments are shown in Figure 2. For the experiments shown in Figure 2, all cells were cultured in a medium that does not contain any nitrogen source so that the cells must be capable of performing nitrogen fixation to grow. All strains show normal growth when cultured in the presence of sucrose (Figures 2E–2H). However, for DJ1445 there is no growth when cells are depleted for IscU (Figure 2B). For strain DJ1445, depletion of IscU eliminates the capacity for growth under nitrogen-fixing conditions (Figure 2B) or when a fixed nitrogen source is added to the growth medium (results not shown). These results suggest that IscU is essential under both growth conditions and that NifU cannot functionally replace IscU.
A possible explanation for the inability of NifU to functionally replace IscU is that NifU is sequestered into a macromolecular nif-specific complex so that it is not available for other cellular processes. To test this possibility, strain DJ1496 was constructed, which contains two copies of the nifU and nifS genes, one copy whose expression is under nif control and the other copy under scr control. This strain was then used as a recipient in genetic transformation experiments where we attempted to delete separately the iscU gene and the iscS gene. In these experiments, neither the iscU gene nor the iscS gene could be deleted, even under conditions where nifU and nifS are expressed independently from the nif-specific components and in the absence of other nif-specific components. To confirm that nifU and nifS are actually expressed when regulated by the scr promoter, a derivative of DJ1496 was constructed, where the nif-regulated copies of nifU and nifS are deleted (Figure 1, strain DJ1475). This strain is capable of growing under nitrogen-fixing conditions when cultured in the presence of sucrose (Figure 2H), but not when cultured in the absence of sucrose (Figure 2D), establishing that the scr-regulated nifU and nifS gene products have functional activity.
Although the specific function of the molecular chaperones, HscB and HscA (Figure 1) is not understood, they are required for isc-directed [Fe-S] cluster assembly and IscU is known to interact specifically with an HscBA complex . This interaction depends on an oligopeptide sequence within IscU (LPPVK), which is necessary and sufficient to stimulate intrinsic HscA-directed ATPase activity . Comparison of IscU and NifU primary sequences shows that the LPPVK signature sequence within IscU is replaced by LPPEK in NifU [13,18]. We therefore considered a second possible explanation for the inability of NifU to replace functionally IscU. Namely, that NifU does not productively interact with the molecular chaperones HscBA, and that such a specific interaction might be required for maturation of [Fe-S] proteins other than the nitrogenase components. To test this possibility, the LPPEK sequence in NifU was converted into the canonical LPPVK sequence within IscU, and experiments similar to those already described were repeated. However, this modification did not endow NifU with an ability to functionally replace IscU. In aggregate, these experiments establish that, under the conditions used here, there is a high degree of specificity for [Fe-S] cluster assembly components in A. vinelandii. In particular, the nif-specific [Fe-S] cluster assembly components are required to maintain an active nitrogenase and cannot be effectively used to replace the isc-specific [Fe-S] cluster assembly components required for the maturation of other [Fe-S] proteins, such as aconitase.
Our results are relevant to a recent report by Takahashi and co-workers where it was shown that heterologous expression of a ‘nif-like’ [Fe-S] cluster biosynthetic system from Entamoeba histolytica could replace the function of the suf- or isc-type of [Fe-S] cluster biosynthetic systems, but only under anaerobic conditions . This finding is in line with the suggestion that nif-like [Fe-S] cluster biosynthetic systems from non-nitrogen-fixing organisms, such as E. histolytica and H. pylori, do not have a specialized function but instead, are utilized for ‘housekeeping’ [Fe-S] protein maturation in these organisms. It therefore appears that, in spite of considerable primary sequence identity among members of nif-like and genuine nif-specific [Fe-S] cluster biosynthetic systems, the nif-specific system (at least for A. vinelandii) has evolved an exquisite function specialized for nitrogenase maturation. We believe that it should be possible to exploit primary sequence differences between components of nif-like and nif-specific [Fe-S] cluster biosynthetic systems, differences in their corresponding three-dimensional structures (when they become available), as well as genetic strategies, to determine the basis for target specificity, a feature that is not yet understood for any [Fe-S] protein maturation process.
This work was supported by the National Science Foundation (MCB-021138).
International Hydrogenases Conference 2004: Independent Meeting held at the University of Reading, 24–29 August 2004. Edited by R. Cammack (King's College London, U.K.) and F. Sargent (University of East Anglia, Norwich, U.K.). Organized by R. Cammack and R. Robson (University of Reading, U.K.). Sponsored by COST (European Cooperation in the field of Scientific and Technical Research), the European Science Foundation and the European Office of Aerospace Research and Development.
- © 2005 The Biochemical Society