Time-resolved Studies of IsdG Protein Identify Molecular Signposts along the Non-canonical Heme Oxygenase Pathway

Abstract

Heme oxygenases (HOs) 3 are enzymes that oxidatively liberate iron from the heme tetrapyrrole (1-3). In the well characterized HOs from animals and many bacteria, the same heme molecule acts as both the O 2 -activating cofactor and substrate. Three successive monooxygenation steps yield Fe(II), CO, and biliverdin IX␣ as the end products of the reaction (Fig. 1) (2, 3). Animals use HOs to maintain cellular heme homeostasis as part of a constant cycle of heme synthesis and breakdown. The products report on the status of this cycle and serve as antioxidants and signaling agents (4, 5). Many bacteria also use HO homologs, both to control heme homeostasis and to liberate iron from host-derived heme (6, 7). Heme, found primarily in hemoglobin, can therefore be used as a rich nutritional source of iron. Because of the intriguing nature of the reaction, which uses heme as both cofactor and substrate (8 -10), as well as the acute biological importance of HO-mediated processes, HOs from several species have been exceptionally well characterized (2, 3).By the early 2000s, however, it was apparent that many important Gram-positive pathogens that degrade host heme did not possess an HO-encoding gene in their genomes. A new family of heme-degrading proteins known as IsdGs was subsequently discovered, with representatives found in bacteria from both Gram-positive and Gram-negative phyla (11). IsdG family proteins are evolutionarily and structurally distinct from the well studied HOs (12, 13), and they yield different end products. Instead of biliverdin IX␣ and CO, the IsdG protein from Mycobacterium tuberculosis (known as MhuD) generates triply-oxygenated linear tetrapyrroles called mycobilins (Fig. 1) (14, 15). A formyl group remains appended to pyrrole ring A or B at the site of macrocycle cleavage, and an oxo group is generated on the pyrrole ring on the opposite side. Notably, no C1 product is released (16).Although homologous to MhuD, the IsdG from Staphylococcus aureus degrades heme to yet a third set of products. The macrocycle is not cleaved at the ␣-meso-but rather at either the ␤-or ␦-meso-carbon. Oxo groups are generated on both the carbon backbone and the pyrrole rings at the cleavage site, generating tetrapyrrole products known as staphylobilins (Fig. 1) (17). It was recently shown that a C1 product is indeed released by the S. aureus IsdG; however, quite unexpectedly, the major C1 product was determined to be formaldehyde (CH 2 O) instead of CO (18). Unlike CO, formaldehyde may be undetectable by animal immune systems, offering a potential selective advantage for heme-feeding pathogens that use IsdG-type enzymes (5,19,20). Mechanistically, the observation that CH 2 O instead of CO implies that verdoheme, the green inter-* This work was supported in part by National Institutes of Health Grants GM090260 and 5P20RR02437 of the CoBRE Program (to J. L. D.) and Grant RO1 AI069233 (to E. P. S.). The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the resp...

Reactions of Ferrous Coproheme Decarboxylase (HemQ) with O2 and H2O2 Yield Ferric Heme b

“…Following the initial hydroxylation, a series of further reactions leads to ring opening and release of formaldehyde and the triply oxygenated linear tetrapyrrole product. 20, 39 …”

Tight binding of heme to Staphylococcus aureus IsdG and IsdI precludes design of a competitive inhibitor

“…6 One difference between these classes of enzymes to potentially target is the first oxygenation reaction catalyzed by both HO families: the conversion of heme to meso-hydroxyheme. 10, 11 Canonical HOs rely upon a conserved water cluster to guide a transient hydroxyl radical to the meso carbon of heme (Fig. 1), 12-14 whereas it has been proposed that non-canonical HOs proceed through a bridged Fe–O–O–C transition state.…”

“…2). 11 Based upon data available prior to this publication, the most promising strategy for selective inhibition of IsdG and IsdI in the presence of human HOs was competitive inhibition since the dissociation equilibrium constants for heme from IsdG and IsdI were reported to be 1000-fold greater than for heme from human HOs. 5, 18, 19 …”

“…A potential explanation for these observations relates to the fact that the K d values for IsdG–heme and IsdI–heme were determined by fitting UV/Vis absorption titration data to a Michaelis-Menten model. 5 Subsequent research has called into question whether this enzyme follows Michaelis-Menten kinetics due to the requirement of at least three substrates, 5, 7, 11 plus a reductase, 22 and the presence of at least two enzymatic intermediates. 11 Thus, the dissociation constants for IsdG–heme and IsdI–heme were re-investigated.…”

Monooxygenase Substrates Mimic Flavin to Catalyze Cofactorless Oxygenations

“…We have compared the properties of the reaction in both the presence and absence of the enzyme to understand how the latter contributes to catalysis. Finally, we have interpreted the results in light of mechanistic work with IsdG (from Staphylococcus aureus) (21) and the emerging model for catalysis by the increasingly well characterized cofactor-independent oxygenase, (1H)-3-hydroxy-4-oxoquinaldine 2,4-dioxygenase (HOD) (4,(22)(23)(24)(25)(26)(27).…”