Spectroscopic Evidence for Electronic Control of Heme Hydroxylation by IsdG.

Staphylococcus aureus IsdG catalyzes a unique trioxygenation of heme to staphylobilin, and the data presented in this article elucidate the mechanism of the novel chemical transformation. More specifically, the roles of the second-sphere Asn and Trp residues in the monooxygenation of ferric-peroxoheme have been clarified via spectroscopic characterization of the ferric-azidoheme analogue. Analysis of UV/vis absorption data quantified the strength of the hydrogen bond that exists between the Asn7 side chain and the azide moiety of ferric-azidoheme. X-band electron paramagnetic resonance data were acquired and analyzed, which revealed that this hydrogen bond weakens the π-donor strength of the azide, resulting in perturbations of the Fe 3d based orbitals. Finally, nuclear magnetic resonance characterization of 13C-enriched samples demonstrated that the Asn7···N3 hydrogen bond triggers partial porphyrin to iron electron transfer, resulting in spin density delocalization onto the heme meso carbons. These spectroscopic experiments were complemented by combined quantum mechanics/molecular mechanics computational modeling, which strongly suggested that the electronic structure changes observed for the N7A variant arose from loss of the Asn7···N3 hydrogen bond as opposed to a decrease in porphyrin ruffling. From these data a fascinating picture emerges where an Asn7···N3 hydrogen bond is communicated through four bonds, resulting in meso carbons with partial cationic radical character that are poised for hydroxylation. This chemistry is not observed in other heme proteins because Asn7 and Trp67 must work in concert to trigger the requisite electronic structure change.

Ruffling is essential for Staphylococcus aureus IsdG-catalyzed degradation of heme to staphylobilin.

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. 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. In general, 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-to­iron 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 spectrometry was used to identify the product distributions, for variants of IsdG with altered substrate conformational distributions. In general, the rate of heme oxygenation by non-canonical heme oxygenases depends 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 population 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.