Summary Enzymatic heme catabolism by heme oxygenases is conserved from bacteria to humans and proceeds through a common mechanism leading to the formation of iron, carbon monoxide, and biliverdin. The first members of a novel class of heme oxygenases were recently identified in Staphylococcus aureus (IsdG and IsdI) and were termed the IsdG-family of heme oxygenases. Enzymes of the IsdG-family form tertiary structures distinct from those of the canonical heme oxygenase family, suggesting that IsdG-family members degrade heme via a unique reaction mechanism. Herein we report that the IsdG-family of heme oxygenases degrade heme to the oxo-bilirubin chromophore staphylobilin. We also present the crystal structure of heme-bound IsdI in which heme ruffling and constrained binding of oxygen is consistent with cleavage of the porphyrin ring at the β– or γ–meso carbons. Combined, these data establish that the IsdG-family of heme oxygenases degrade heme to a novel chromophore distinct from biliverdin.

IsdI, a heme-degrading protein from Staphylococcus aureus , binds heme in a manner that distorts the normally planar heme prosthetic group to an extent greater than that observed so far for any other heme-binding protein. To understand better the relationship between this distinct structural characteristic and the functional properties of IsdI, spectroscopic, electrochemical, and crystallographic results are reported that provide evidence that this heme ruffling is essential to the catalytic activity of the protein and eliminates the need for the water cluster in the distal heme pocket that is essential for the activity of classical heme oxygenases. The lack of heme orientational disorder in 1 H-NMR spectra of the protein argues that the catalytic formation of β- and δ-biliverdin in nearly equal yield results from the ability of the protein to attack opposite sides of the heme ring rather than from binding of the heme substrate in two alternative orientations.

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Background: Heme bound to the heme degrading enzyme IsdI is distorted in the form of ruffling.Results: IsdI Trp66 variants are less catalytically active, and heme bound to the W66Y variant is less distorted.Conclusion: Extensive heme ruffling is required for optimal catalytic activity of IsdI.Significance: The contribution of heme ruffling to catalysis of heme oxidation is a feature that distinguishes IsdI-like enzymes from heme oxygenase.

IsdG and IsdI are paralogous proteins from Staphylococcus aureus that reductively degrade hemin. Final heme degradation products of IsdG and IsdI are yet to be elucidated, nor has it been determined how oxygen binds to initiate the reaction. The crystal structures of an inactive N7A variant of IsdG in complex with Fe 3+ -protoporphyrin IX (IsdG-hemin) and of IsdI in complex with cobalt protoporphyrin IX (IsdI-CoPPIX) were solved to 1.8 Å or better resolution. These structures show that the metalloporphyrins are buried into similar deep clefts and the propionic acids form salt bridges to two Arg residues. His77 (IsdG) or His76 (IsdI), a residue required for activity, is coordinated to Fe 3+ or Co 3+ atoms, respectively. The bound porphyrin rings form extensive steric interactions in the binding cleft such that the porphyrin rings are highly distorted from the planar. This distortion can be described as ruffled and places the b-and d-meso carbons close to the distal oxygen-binding site. In the structure of IsdI-CoPPIX, the distal side of the CoPPIX accommodates a chloride ion in a cavity formed through a conformational change in Ile55. The chloride ion participates in a hydrogen bond to the side chain amide of Asn6. We also have determined the crystal structures of IsdG-hemin and IsdI-hemin bound to cyanide to resolutions of ~1.8 Å. Structural information from these complexes is valuable in understanding the regiospecificity of ring cleavage. We propose a reaction mechanism in which reactive peroxide intermediate proceeds with nucleophilic oxidation at the b-or d-meso carbon of the hemin.Keywords: iron acquisition, heme degradation, staphylococcus aureus P04.03.199 Acta Cryst. (2008). A64, C293Crystal structural analysis of photosystem II with the novel method to reduce X-ray radiation damage Photosystem II (PSII) is a multi-subunit membrane protein complex functioning in photosynthesis. It performs a series of light-induced electron transfer reactions leading to the splitting of water and generation of molecular oxygen. The catalytic center is composed of four manganese atoms and one calcium atom. The detailed locations of these metal atoms are not fully understood. Recent X-ray absorption fine structure analysis has shown that the Mn4Ca-cluster is seriously damaged by X-ray irradiation onto PSII crystals resulting in changes in the coordination structure among these metal atoms. In order to reveal the intact structure of the Mn4Ca-cluster, we adopted a slide-oscillation method to collect the low-dose X-ray data for the crystal structure analysis. In this method, the irradiation point was slid to an adjacent point on the crystal along the oscillation axis after recording of every image and after some slides the irradiation point was brought back to the initial position. These slides were repeated through the required oscillation range. This method reduced the overlap of the irradiated regions in each cross section of the crystal 20 times lower than the normal oscillation method. Using X-ray beam (0.035 x 0.035 mm...