DATA COLLECTION FROM SMALL CRYSTALS OF LARGE ENZYME ACONITASE

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Crystallographic experiments with mitochondrial (mAc) are providing insight into the active site structure, substrate binding and catalytic mechanism of this [Fe-S] enzyme. Application of the mutagenesis and expression methods we have developed for mAc is allowing fundamental questions about [Fe-S] cluster chemistry in proteins to be approached through substrate and inhibitor binding and cluster design and engineering experiments. On-going structural studies of a 7Fe ferredoxin (FdI) are defining the factors controlling [Fe-S] cluster structure and properties and providing insight into the chemical and conformational behavior of inorganic [Fe-S] cores in a protein environment. mAc is an 83 kD protein containing a [4Fe-4S] cluster, which participates directly in catalysis. FdI is a 13 kD electron transfer protein containing [3Fe-4S] and [4Fe-4S] clusters. Crystals of recombinant aconitase diffract to 1.8-2.0 E resolution at 100 K. However, they can not be grown larger that 0.3 x 0.1 x 0.05 mm. In order to study the critical details of the active site, the binding of small molecules and the precise geometry of [Fe-S] clusters, it is necessary to acquire data to at least 2.5 E and preferably to 2.0-1.8 E. For mAc crystals this is only possible with synchrotron radiation. FdI crystals are large and diffract very well. However, in order to carry out full matrix least squares refinement of the [Fe-S] clusters, it is necessary to collect data to 1.4 E resolution, and this also requires synchrotron radiation.