Ruffling is Essential for Staphylococcus aureus IsdG-catalyzed Degradation of Heme to Staphylobilin

Abstract

Non-canonical heme oxygenases are enzymes that degrade heme to non-biliverdin products within bacterial heme iron acquisition pathways. These enzymes all contain a conserved second-sphere Trp residue that is essential for enzymatic turnover. Previous studies have revealed several important roles for the conserved second-sphere Trp in Staphylococcus aureus IsdG, S. aureus IsdI, and Mycobacterium tuberculosis MhuD. However, a general model for the geometric, electron-ic, and functional role of the second-sphere Trp had not been deduced prior to this work. Here, UV/Vis absorption (Abs) and circular dichroism (CD) spectroscopies were employed to show that the W67F variant of IsdG perturbs the heme substrate conformation without altering the protein secondary structure. In general, it can now be stated that a dynamic equilibrium between “planar” and “ruffled” substrate conformations exists within non-canonical heme oxygenases, and that the second-sphere Trp favors population of the “ruffled” substrate conformation. 1H nuclear magnetic resonance and magnetic CD spectroscopies were used to characterize the electronic structures of IsdG and IsdI variants with different substrate conformational distributions. These data revealed that the “ruffled” substrate conformation promotes partial porphyrin-to-iron electron transfer, which makes the meso carbons of the porphyrin ring susceptible to radical attack. Finally, UV/Vis Abs spectroscopy was utilized to quantify the enzymatic rates, and electrospray ionization mass spec-trometry was used to identify the product distributions, for variants of IsdG with altered substrate conformational distri-butions. In general, the rate of heme monooxygenation to meso-hydroxyheme by non-canonical heme oxygenasess de-pends upon the population of the “ruffled” substrate conformation. Also, the production of staphylobilin or mycobilin by these enzymes is correlated with the population of the “ruffled” substrate conformation, since variants that favor popula-tion of the “planar” substrate conformation yield significant amounts of biliverdin. These data can be understood within the framework of a concerted rearrangement mechanism for the monooxygenation of heme to meso-hydroxyheme by non-canonical heme oxygenases. However, the mechanisms of IsdG/IsdI and MhuD must diverge following this interme-diate in order to generate distinct staphylobilin and mycobilin products, respectively.

Stabilization of the Ferryl=oxoheme Form of Staphylococcus aureus IsdG by Electron Transfer from a Second-Sphere Tryptophan

“…23,31 Previous studies of analogues for the ferric-peroxoheme "reactant" species have revealed that two second-sphere amino acids, Asn7 and Trp67, work in concert to control the regiochemistry of the reaction by orienting the peroxo ligand and distorting the porphyrin ligand. 25,[45][46][47][48] However, analogues for the hydroxylated fer-ryl=oxoheme "product" species have not been characterized in-depth. A stable ferryl=oxoheme form of IsdG would be an excellent analogue for the this "product" species, and its characterization is expected to yield critical insight into the rate-limiting step of IsdG-catalyzed heme degration.…”

Regulatory mechanism of formaldehyde release in heme degradation catalyzed by Staphylococcus aureus IsdG

“…Because the unusual reaction modes can be attributed to the hydroxyheme ruffling, the mechanism underlying the activity of IsdG and IsdI is thought to be similar to that underlying the activity of MhuD, despite the formation of different products. Recent mass studies have detected a new product in the IsdG reactions ( 20 , 21 ). The reports claim this product to be SB retaining a formyl group at the cleavage site, termed “formyl-SB” in this manuscript ( Fig.…”