C-13 NMR spectroscopic studies have been conducted with the hydroxide complex of Pseudomonas aeruginosa heme oxygenase (Fe-III-OH), where OH- has been used as a model of the OOH- ligand to gain insights regarding the elusive ferric hydroperoxide (Fe-III-OOH) intermediate in heme catabolism at ambient temperatures. Analysis of the heme core carbon resonances revealed that the coordination of hydroxide in the distal site of the enzyme results in the formation of at least three populations of Fe-III-OH complexes with distinct electronic configurations and nonplanar ring distortions that are in slow exchange relative to the NMR time scale. The most abundant population exhibits a spin crossover between S = 1/2 and S = 3/2 spin states, and the two less abundant populations exhibit pure, S = 3/2 and S = 1/2, (d(xy))(1) electronic configurations. We propose that the highly organized network of water molecules in the distal pocket of heme oxygenase, by virtue of donating a hydrogen bond to the coordinated hydroxide ligand, lowers its ligand field strength, thereby increasing the field strength of the porphyrin (equatorial) ligand, which results in nonplanar deformations of the macrocycle. This tendency to deform from planarity, which is imparted by the ligand field strength of the coordinated OH-, is likely reinforced by the flexibility of the distal pocket in HO. These findings suggest that if the ligand field strength of the coordinated OOH- in heme oxygenase is modulated in a similar manner, the resultant large spin density at the meso carbons and nonplanar deformations of the pophyrin ring prime the macrocycle to actively participate in its own hydroxylation.