Reaction intermediates and single turnover rate constants for the oxidation of heme by human heme oxygenase-1

Heme oxygenase converts heme to biliverdin, iron, and CO in a reaction with two established intermediates, alpha-meso-hydroxyheme and verdoheme. Transient kinetic studies show that the conversion of Fe3+-heme to Fe3+-verdoheme is biphasic. Electron transfer to the heme (0.11 s(-1) at 4 degrees C and 0.49 s(-1) at 25 degrees C) followed by rapid O-2 binding yields the ferrous dioxy complex. Transfer of an electron (0.056 s(-1) at 4 degrees C and 0.21 s(-1) at 25 degrees C) to this complex triggers the formation of alpha-meso-hydroxyheme and its subsequent O-2-dependent fragmentation to Fe3+-verdoheme. The conversion of Fe3+ verdoheme to Fe3+-biliverdin is also biphasic. Thus, reduction of Fe3+ to Fe2+-verdoheme (0.15 s(-1) at 4 degrees C and 0.55 s(-1) at 25 degrees C) followed by O-2 binding and an electron transfer produces Fe3+-biliverdin (0.025 s(-1) at 4 degrees C and 0.10 s(-1) at 25 degrees C). The conversion of Fe3+-biliverdin to free biliverdin is triphasic. Reduction of Fe3+-biliverdin (0.035 s(-1) at 4 degrees C and 0.15 s(-1) at 25 degrees C), followed by rapid release of Fe2+ (0.19 s(-1) at 4 degrees C and 0.39 s(-1) at 25 degrees C), yields the biliverdin-enzyme complex from which biliverdin slowly dissociates (0.007 s(-1) at 4 degrees C and 0.03 s(-1) at 25 degrees C). The rate of Fe2+ release agrees with the rate of Fe3+-biliverdin reduction. Fe2+ release clearly precedes biliverdin dissociation. In the absence of biliverdin reductase, biliverdin release is the rate-limiting step, but in its presence biliverdin release is accelerated and the overall rate of heme degradation is limited by the conversion of Fe2+-verdoheme to the Fe3+-biliverdin.