Catalytic mechanism

The reaction mechanism for APS reductase was based on a nucleophilic attack of the N5 atom of reduced FAD on the sulfur of APS involving FAD-APS and FAD-sulfite intermediates originally postulated by Michaels (Michaels et al., 1970). The recently reported crystal structure of APS reductase (Fritz et al., 2002b) confirmed this hypothesis and a structure-based mechanism was outlined.

Reduction of FAD to FADH2 and subsequent binding of APS initiated the reaction cycle.

Atom N5 of FADH2 attacked the sulfur of the APS to form a FAD-APS adduct. The proposed intermediate decomposed spontaneously to AMP and to the FAD-sulfite adduct, and sulfite became liberated. Presumably, the key step in the reaction cycle was the formation of the

FAD-APS intermediate which was facilitated if the atom N5 of FAD became more nucleophilic, and the sulfate sulfur more electrophilic. Furthermore, this first step could be driven through electrostatic stabilization of the negatively charged FAD-APS intermediate by the surrounding polypeptide matrix.

On the basis of the structures of several intermediate states described in this work a more details on the mechanism could be given.

The binding of APS was optimal for a nucleophilic attack by the N5 nitrogen of reduced FAD on the sulfur of APS (Figure 4.4B). The distance between the sulfur and N5 of FAD was about 3.6 Å, which corresponded to van der Waals contact. A striking observation was that upon APS binding the isoalloxazine ring was pushed backwards to avoid interference with the bound sulfate group of APS. The strained conformation of FAD increased the energy of the substrate complex, which in turn reduced the activation energy of the reaction. In order to form the flavin-APS adduct (Figure 4.4C) the FAD had to swing even more back than observed in the APSR-d-red state (Figure 4.4A) to optimize molecular orbital overlap.

Interestingly, the oxygens of the sulfate of APS and the oxygens of the sulfite adduct (Figure 4.4E) were in close proximity. This suggested that during covalent binding the sulfur moved towards N5 under inversion of the configuration of the oxygens. The shift of the sulfur of around 1 Å towards N5 probably did not cause a large shift in the AMP part of APS such that its binding mode was maintained.

The electrophilicity of the sulfur was increased by the formation of hydrogen bonds between the sulfate oxygens and Asn A74, Arg A265 and His A389. The importance of these residues was confirmed by strict conservation in all known APS reductases (data not shown). The nucleophilicity of the N5 atom of FAD was enhanced as a consequence of the deprotonation of the atom N1 in the APSR-red state. The resulting negative charge became primarily delocalized over the N1-C=O2 group (Ghisla & Massey, 1986) but also over the entire isoalloxazine ring including atom N5. The counterbalancing positive charge necessary to maintain the unprotonated state was provided by two hydrogen bonds donated from the polypeptide to the O2 atom of FAD and by the large dipole of a 30 Å long helix, that was pointing with its N-terminus directly towards the N1 atom.

N^- O

Figure 4.5: Structure based reaction mechanism of APS reductase from A. fulgidus. It was based on the structures of APS reductase in complex with APS, AMP+sulfite and sulfite as well as the structures of the oxidized and reduced substrate free enzyme.

The formation of the FAD-APS intermediate (Figure 4.4C) was accompanied by the deprotonation of the N5 hydrogen. The fate of this proton couldn’t be followed directly because the structures did not provide an unambiguous answer. The nucleophilic attack took place on the re-side of FAD with the proton located on its si-side. The si-side of FAD was rather hydrophobic and the only possible acceptor was a water molecule that was too far away (6.7 Å). Thus, the most likely scenario was a proton transfer from the si-side of FAD to the

O2B of APS concomitant with the nucleophilic attack. For the subsequent transfer of the proton three pathways were conceivable:

(i) The proton could be located on a sulfite oxygen during the reaction cycle. This is very unlikely as the pKA of FAD-sulfite adducts is very low.

(ii) His A398 could be an acceptor of a proton localized on O2B. The prerequisite, however, was that its NE2 atom was not protonated. This might have been the case in substrate free enzyme as the hydrophobic environment obstructed protonation of His A398. Its ND1 atom was not in hydrogen-bonding distance to Ser A399 before but upon binding of APS it was hydrogen bonded to the NH group of Ser A399. In the APSR-amp state it was also hydrogen bonded to the Ser A399 OH group so the histidinyl proton needed to be located on the NE2 atom. The double hydrogen bonding together with the short His NE2 FAD-sulfite O2 distance suggested a different role for His A398: the stabilization of the negative charge on the sulfite adduct in the APSR-amp state.

(iii) The proton could be transferred via hydrogen bonds from oxygen O2B of the sulfate group to water 5621 and then to water 5422. The positive charge on water 5422 could be stabilized by hydrogen bonding to OE1 and OE2 of Glu A141 and OD1 of Asn A74. The only drawback was that Glu A141 was only conserved in some APS reductases (data not shown).

In the next step the formed flavin-APS adduct was cleaved resulting in a flavin-sulfite adduct and AMP, the S-O bond of the phosphosulfate anhydride being instable and cleaved spontaneously. The twice negatively charged phosphate group of the released AMP was shifted towards Arg A265 to increase the distance to the sulfite and to be optimally hydrogen bonded compensating those charges. Simultaneously, the sulfite rotated in order to minimize the interactions with the AMP and optimized the charge compensation by His A389. The repulsion between the negative charge of the sulfite and the AMP might have facilitated the release of AMP. However, the positive environment needed to be able to compensate these charges as the enzyme also catalyzed the back reaction. This was mainly achieved by the strong bidentate salt bridge to Arg A265.

After AMP cleavage the sulfite of the FAD-sulfite adduct rotated back resulting in different hydrogen bonding to the protein. The longer “protein” - sulfite distances reflecting the protonation of the sulfite and facilitating the FAD-sulfite bond cleavage.

In the final step the sulfite was cleaved from FAD. This reaction was accelerated by protonating the sulfite via the activated water molecules.

With the leaving of the product hydrogen-sulfite the FAD rotated back into the original position the surrounding residues adjusting to this. The flavin was in the oxidized state and needed to be reduced for the next reaction cycle.