Oxidation of heme to β- and δ-biliverdin by Pseudomonas aeruginosa heme oxygenase as a consequence of an unusual seating of the heme

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The origin of the unusual regioselectivity of heme oxygenation, i.e. the oxidation of heme to δ-biliverdin (70%) and β-biliverdin (30%), that is exhibited by heme oxygenase from Pseudomonas aeruginosa (pa-HO) has been studied by 1H NMR, 13C NMR, and resonance Raman spectroscopies. Whereas resonance Raman indicates that the heme-iron ligation in pa-HO is homologous to that observed in previously studied α-hydroxylating heme oxygenases, the NMR spectroscopic studies suggest that the heme in this enzyme is seated in a manner that is distinct from that observed for all other α-hydroxylating heme oxygenase enzymes for which a structure is known. In pa-HO, the heme is rotated in-plane ∼ 110°, so the δ-meso-carbon of the major orientational isomer is located within the HO-fold in the place where the α-hydroxylating enzymes typically place the α-meso-carbon. The unusual heme seating displayed by pa- HO places the heme propionates so that these groups point in the direction of the solvent-exposed heme edge and appears to originate in large part from the absence of stabilizing interactions between the polypeptide and the heme propionates, which are typically found in α-hydroxylating heme oxygenase enzymes. These interactions typically involve Lys-16 and Tyr-112, in Neisseriae meningitidis HO, and Lys-16 and Tyr-134, in human and rat HO-1. The corresponding residues in pa-HO are Asn-19 and Phe-117, respectively. In agreement with this hypothesis, we found that the Asn-19 Lys/Phe-117 Tyr double mutant of pa-HO exists as a mixture of molecules exhibiting two distinct heme seatings; one seating is identical to that exhibited by wild-type pa-HO, whereas the alternative seating is very similar to that typical of α-hydroxylating heme oxygenase enzymes and is related to the wild-type seating by ∼ 110° in-plane rotation of the heme. Furthermore, each of these heme seatings in the pa-HO double mutant gives rise to a subset of two heme isomeric orientations that are related to each other by 180° rotation about the α-γ-mesoaxis. The coexistence of these molecules in solution, in the proportions suggested by the corresponding area under the peaks in the 1H NMR spectrum, explains the unusual regioselectivity of heme oxygenation observed with the double mutant, which we found produces α- (55%), δ- (35%), and β-biliverdin (10%). α-Biliverdin is obtained by oxidation of the heme seated similar to that of α-hydroxylating enzymes, whereas β- and δ-biliverdin are formed from the oxidation of heme seated as in wild-type pa-HO.

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Journal of the American Chemical Society

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