Aconitase cluster interconversion

By this time it was demonstrated that the [3Fe-4S]W+ form of aconitase is inactive, while the [4Fe-4S]2+ form is active. How is the activity of the enzyme affected by the oxidation state of the [4Fe-4S] cluster Because the active enzyme contains a [4Fe-4S]2+ cluster, either the 3+ or 1+ oxidation states may also be stable. The 3+ state is unstable since oxidation of the [4Fe-4S]2+ resulted in the immediate loss of a ferrous ion and conversion to a [3Fe-4S]i+ cluster (46,47). However, reduction of active aconitase by sodium dithionite or photoreduction in the presence of deazaflavin produced in high yields an EPR signal characteristic for [4Fe-4S]l+ clusters (47). When active enzyme within an anaerobic assay cuvette was photoreduced, the activity of the enzyme dropped to 1/3 of its initial value. Further photoreduction resulted in cluster destruction. Then, if the enzyme is reoxidized with air, the activity returned to its original value. This demonstrated that the redox state of the cluster can modulate the enzyme activity. A scheme summarizing the cluster interconversions and various redox states of the Fe-S cluster of aconitase is shown below. 

Why did nature use an Fe-S cluster to catalyze this reaction, when an enzyme such as fumarase can catalyze the same type of chemistry in the absence of any metals or other cofactors One speculation would be that since aconitase must catalyze both hydrations and dehydrations, and bind substrate in two orientations, Fe in the comer of a cubane cluster may provide the proper coordination geometry and electronics to do all of these reactions. Another possibility is that the cluster interconversion is utilized in vivo to regulate enzyme activity, and thus, help control cellular levels of citrate. A third, but less likely, explanation is that during evolution an ancestral Fe-S protein, whose primary function was electron transfer, gained the ability to catalyze the aconitase reaction through random mutation. 

Figure 9. Schematic representation of cluster interconversion reactions of aconitase and Dg Fd II. Mdssbauer spectroscopic parameters refer to [4Fe-4S] clusters (124, 132) ox = oxidant. With aconitase, subsite a is occupied by Fe upon cluster conversion, whereas with Dg Fd II one or more of the subsites b are populated in this process.

The [3Fe-4S]/[4Fe-4S] cluster interconversion of aconitase is clearly subsite-specific because one and the same site is involved in the two reactions. The similar interconversion of Dg Fd II may be equally subsite-specific, but this cannot be proven. In this case, the three subsites b of the [4Fe-4S] cluster cannot actually be equivalent but are indistinguishable by the technique of resolution at hand, Mossbauer spectroscopy. What is... 

Fig. 17. Interconversion of a [4Fe-4S] cluster to a [4Fe-3S] cluster in the active site of aconitase Fe refers to enriched 56Fe and 57Fe (42).

The beef heart enzyme (M, = 80,000) (117) is a component of the citric acid cycle. Its active form contains one [4Fe-4S] cluster. Although such a cluster is normally associated with electron transfer, the enzyme catalyzes the nonredox reaction of citrate-isocitrate interconversion via a dehydration-hydration pathway. The current state of understanding of cluster structures and reactions of beef heart aconitase has been thoroughly reviewed by Emptage (130). When isolated aerobically, aconitase is inactive and contains one [3Fe-4S] cluster. Upon incubation of the reduced protein with Fe(ll), the fully active enzyme is generated. When a 3-Fe center is reduced to [3Fe-4S]°, Reaction 10 builds a 4-Fe cluster in a nonredox process. The Mossbauer spectra in Fig. 8 address the question of subsite specificity in this reaction of aconitase (124). If the externally supplied iron is Fe, the resultant spectrum reveals the intrinsic (original) Fe atoms... 

The [3Fe-4S] clusters may be regarded as being derived from [4Fe-4S] with one Fe subsite vacant [170-172]. Only two oxidation levels have so far been identified. These are the 1 -I- level, which gives a distinctive EPR spectrum (S = 1/2 and gav > 2), and the 0 level (S = 2). In some instances, for example aconitase, interconversion between [3Fe-4S] and [4Fe-4S] clusters—associated with the inactive and active enzyme, respectively—is known to occur readily [173-175]. 

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