Labile cluster

Aconitase was first suggested to have an iron-sulfur cluster by virtue of the presence of acid-labile sulfide, as well as a g = 2.01 EPR signal, in the purified protein. The iron-sulfur cluster giving rise to this g = 2.01 signal was later shown to be a [3Fe-4S]" " cluster, and was characteristic of inactive enzyme. " " Activation by addition of iron and reductant, or by addition of reductant alone, provided the enzymatically active aconitase, although in the latter case only approximately 75% of full activity was obtained. As the active form of aconitase was subsequently shown to be the [4Fe-4S] form, " addition of iron and reductant provided the iron needed to occupy the fourth metal site in the cluster, while addition of reductant alone resulted in rearrangement of the clusters, labilizing iron to allow reassembly of [4Fe-4S] clusters in 3/4 of the cluster sites. 

In summary, the common CX2CX3C cluster-binding motif found in the radical-SAM enzymes confers some common properties to the clusters in these enzymes, including cluster lability. However, the details of the lability and the precise cluster forms observed vary from enzyme to enzyme. 

The efficiency of separation of solvent from solute varies with their nature and the rate of flow of liquid from the HPLC into the interface. Volatile solvents like hexane can be evaporated quickly and tend not to form large clusters, and therefore rates of flow of about 1 ml/min can be accepted from the HPLC apparatus. For less-volatile solvents like water, evaporation is slower, clusters are less easily broken down, and maximum flow rates are about 0.1-0.5 ml/min. Because separation of solvent from solute depends on relative volatilities and rates of diffusion, the greater the molecular mass difference between them, the better is the efficiency of separation. Generally, HPLC is used for substances that are nonvolatile or are thermally labile, as they would otherwise be analyzed by the practically simpler GC method the nonvolatile substances usually have molecular masses considerably larger than those of commonly used HPLC solvents, so separation is good. 

Fig. 4. Representation of the ligand sphere of the [2Fe-2S] cluster of the Rieske protein from spinach and the attribution of g-tensor to moleculEir axes as discussed in the text. Ser 130 has been observed to influence the redox potentiEd of the cluster via hydrogen interactions with the acid-labile bridging sulfur.

Fig. 1. Iron-sulfur clusters basic building blocks. In most cases the iron is tetrahe-drally coordinated by sulfur from cysteinyl residues (and labile sulfur). Variability on coordination is allowed (see text). A, Rubredoxin type FeS4 (simplest cluster, no labile sulfur) B, plant-type ferredoxin [2Fe-2S] C, bacterial ferredoxin [3Fe-4S] D, bacterial ferredoxin and HiPIP [4Fe-4S] E, novel cluster [4Fe-2S, 20] ( hybrid cluster ).

The chlorate reductase has been characterized in strain GR-1 where it was found in the periplasm, is oxygen-sensitive, and contains molybdenum, and both [3Fe-4S] and [4Fe-4S] clusters (Kengen et al. 1999). The arsenate reductase from Chrysiogenes arsenatis contains Mo, Fe, and acid-labile S (Krafft and Macy 1998), and the reductase from Thauera selenatis that is specific for selenate, is located in the periplasmic space, and contains Mo, Fe, acid-labile S, and cytochrome b (Schroeder et al. 1997). In contrast, the membrane-bound selenate reductase from Enterobacter cloacae SLDla-1 that cannot function as an electron acceptor under anaerobic conditions contains Mo and Fe and is distinct from nitrate reductase (Ridley et al. 2006). 

Recently Jensen and co-workers have determined the structure of a clostridial-type ferredoxin obtained from Micrococcus aerogenes (47). One of the two apparently identical iron-sulfur clusters is illustrated in Fig. 2. The structure is compatible with a model with iron and labile sulfide at alternate comers of a cube. This accounts for the equivalence of these moieties in the protein. Another 8-iron-8 labile sulfur ferredoxin, from Clostridium acidiurici, similarly contains two independent iron-sulfur clusters per molecule (48). Strahs and Kraut (49) had earlier discovered... 

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