Active-Site Controlled, Jahn-Teller Enabled Regioselectivity in Reductive S-C Bond Cleavage of S-Adenosylmethionine in Radical SAM Enzymes.

Catalysis by canonical radical S-adenosyl-l-methionine (SAM) enzymes involves electron transfer (ET) from [4Fe-4S]+ to SAM, generating an R3S0 radical that undergoes regioselective homolytic reductive cleavage of the S-C5' bond to generate the 5'-dAdo· radical. However, cryogenic photoinduced S-C bond cleavage has regioselectively yielded either 5'-dAdo· or ·CH3, and indeed, each of the three SAM S-C bonds can be regioselectively cleaved in an RS enzyme. This diversity highlights a longstanding central question: what controls regioselective homolytic S-C bond cleavage upon SAM reduction? We here provide an unexpected answer, founded on our observation that photoinduced S-C bond cleavage in multiple canonical RS enzymes reveals two enzyme classes: in one, photolysis forms 5'-dAdo·, and in another it forms ·CH3. The identity of the cleaved S-C bond correlates with SAM ribose conformation but not with positioning and orientation of the sulfonium center relative to the [4Fe-4S] cluster. We have recognized the reduced-SAM R3S0 radical is a (2E) state with its antibonding unpaired electron in an orbital doublet, which renders R3S0 Jahn-Teller (JT)-active and therefore subject to vibronically induced distortion. Active-site forces induce a JT distortion that localizes the odd electron in a single priority S-C antibond, which undergoes regioselective cleavage. In photolytic cleavage those forces act through control of the ribose conformation and are transmitted to the sulfur via the S-C5' bond, but during catalysis thermally induced conformational changes that enable ET from a cluster iron generate dominant additional forces that specifically select S-C5' for cleavage. This motion also can explain how 5'-dAdo· subsequently forms the organometallic intermediate Ω.

Determination of a novel structure by a combination of long-wavelength sulfur phasing and radiation-damage-induced phasing.

The structure of the 115 amino-acid residue protein DsvC was determined based on the anomalous scattering provided by the five S atoms present in the structure. By collecting the diffraction data at a wavelength of 1.9 A, the anomalous signal provided by the S atoms was enhanced. However, significant radiation damage occurred during the course of the experiment, which led to differences between different parts of the data set. Only by dividing the total data set into five data sets was it possible to obtain phases; these could then be successfully extended to allow structure determination by the automated model-building program ARP/wARP. A computational correction for the radiation damage was found to significantly improve the success rate in determining the heavy-atom substructure and to improve phasing and refinement statistics.

Radiation inactivation analysis of enzymes. Effect of free radical scavengers on apparent target sizes.

In most cases the apparent target size obtained by radiation inactivation analysis corresponds to the subunit size or to the size of a multimeric complex. In this report, we examined whether the larger than expected target sizes of some enzymes could be due to secondary effects of free radicals. To test this proposal we carried out radiation inactivation analysis on Escherichia coli DNA polymerase I, Torula yeast glucose-6-phosphate dehydrogenase, Chlorella vulgaris nitrate reductase, and chicken liver sulfite oxidase in the presence and absence of free radical scavengers (benzoic acid and mannitol). In the presence of free radical scavengers, inactivation curves are shifted toward higher radiation doses. Plots of scavenger concentration versus enzyme activity showed that the protective effect of benzoic acid reached a maximum at 25 mM then declined. Mannitol alone had little effect, but appeared to broaden the maximum protective range of benzoic acid relative to concentration. The apparent target size of the polymerase activity of DNA polymerase I in the presence of free radical scavengers was about 40% of that observed in the absence of these agents. This is considerably less than the minimum polypeptide size and may reflect the actual size of the polymerase functional domain. Similar effects, but of lesser magnitude, were observed for glucose-6-phosphate dehydrogenase, nitrate reductase, and sulfite oxidase. These results suggest that secondary damage due to free radicals generated in the local environment as a result of ionizing radiation can influence the apparent target size obtained by this method.

Radiation inactivation of hepatic sulfite oxidase.

Sulfite oxidase (EC 1.8.3.1), purified from chicken liver, is comprised of two identical subunits of 55 kDa, each of which contains a molybdenum and heme prosthetic group. The functional size of sulfite oxidase was determined by radiation inactivation analysis using both full, sulfite:cytochrome c reductase, and partial, sulfite:ferricyanide reductase, catalytic activities. Inactivation of full enzyme activity indicated a target size of 42 kDa while the partial activity indicated a target size of 25 kDa. These results confirm the earlier findings of two equivalent subunits and suggest the presence of a functional domain within the subunit structure that contains the molybdenum center and exhibits a smaller molecular mass than that of the enzyme subunit.