| Abstract
| - The Escherichia coli DNA repair enzyme MutY plays an important role in the recognitionand repair of 7,8-dihydro-8-oxo-2‘-deoxyguanosine−2‘-deoxyadenosine (OG·A) mismatches in DNA. MutYprevents DNA mutations caused by the misincorporation of A opposite OG by catalyzing thedeglycosylation of the aberrant adenine. MutY is representative of a unique subfamily of DNA repairenzymes that also contain a [4Fe-4S]2+ cluster, which has been implicated in substrate recognition.Previously, we have used site-directed mutagenesis to individually replace the cysteine ligands to the[4Fe-4S]2+ cluster of E. coli MutY with serine, histidine, or alanine. These experiments suggested thathistidine coordination to the iron−sulfur cluster may be accommodated in MutY at position 199. Purificationand enzymatic analysis of C199H and C199S forms indicated that these forms behave nearly identical tothe WT enzyme. Furthermore, introduction of the C199H mutation in a truncated form of MutY (C199HT)allowed for crystallization and structural characterization of the modified [4Fe-4S] cluster coordination.The C199HT structure showed that histidine coordinated to the iron cluster although comparison to thestructure of the WT truncated enzyme indicated that the occupancy of iron at the modified position hadbeen reduced to 60%. Electron paramagnetic resonance (EPR) spectroscopy on samples of C199HTindicates that a significant percentage (15−30%) of iron clusters were of the [3Fe-4S]1+ form. Oxidationof the C199HT enzyme with ferricyanide increases the amount of the 3Fe cluster by approximately 2-fold.Detailed kinetic analysis on samples containing a mixture of [3Fe-4S]1+ and [4Fe-4S]2+ forms indicatedthat the reactivity of the [3Fe-4S]1+ C199HT enzyme does not differ significantly from that of the WTtruncated enzyme. The relative resistance of the [4Fe-4S]2+ cluster toward oxidation, as well as the retentionof activity of the [3Fe-4S]1+ form, may be an important aspect of the role of MutY in repair of DNAdamage resulting from oxidative stress.
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