The origin of the unusual regioselectivity of heme oxygenation, i.e. the oxidation of heme to delta-biliverdin (70%) and beta-biliverdin (30%), that is exhibited by heme oxygenase from Pseudomonas aeruginosa (pa-HO) has been studied by H-1 NMR, C-13 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 a-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 a-hydroxylating heme oxygenase enzymes for which a structure is known. In pa-HO, the heme is rotated in-plane similar to110degrees, so the delta-meso-carbon of the major orientational isomer is located within the HO-fold in the place where the a-hydroxylating enzymes typically place the alpha-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 alpha-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 alpha-hydroxylating heme oxygenase enzymes and is related to the wild-type seating by similar to110degrees 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 180degrees rotation about the alpha-gamma-meso-axis. The coexistence of these molecules in solution, in the proportions suggested by the corresponding area under the peaks in the H-1 NMR spectrum, explains the unusual regioselectivity of heme oxygenation observed with the double mutant, which we found produces alpha- (55%), delta-(35%), and beta-biliverdin (10%). a-Biliverdin is obtained by oxidation of the heme seated similar to that of a-hydroxylating enzymes, whereas beta- and delta-biliverdin are formed from the oxidation of heme seated as in wild-type pa-HO.