A role for highly conserved carboxylate, aspartate-140, in oxygen activation and heme degradation by heme oxygenase-1

Heme oxygenase (HO) catalyzes the oxygen-dependent degradation of heme to b iliverdinIX alpha, CO, and free iron ion via three sequential monooxygenase reactions. Although the distinct active-site structure of HO from cytochro me P450 families suggests unique distal protein machinery to activate molec ular oxygen, the mechanism and the key amino acid for the oxygen activation have not been clear. To investigate the functionality of highly conserved polar amino acids in the distal helix of HO-1, we have prepared alanine mut ants: T135A, R136A, D140A, and S142A, and found drastic changes in the heme degradation reactions of D140A. In this paper, we report the first evidenc e that D140 is involved in the oxygen activation mechanism in HO-1. The hem e complexes of HO mutants examined in this study fold and bind heme normall y. The pK(a) values of the iron-bound water acid autoxidation rates of the oxy-form are increased with R136A, D140A, and S142A mutations, but are not changed with T135A mutation. As the wild-type, T135A, R136A, and S142A degr ade heme to verdohemeIX alpha with H2O2 and to biliverdinIXa with the NADPH reductase system. On the other hand, D140A heme complex forms compound II with H2O2, and no heme degradation occurs. For the NADPH reductase system, the oxy-form of D140A heme complex is accumulated in the reaction, and only 50% of heme is degraded. The stopped flow experiments suggest that D140A c annot activate iron-bound dioxygen and hydroperoxide properly. To investiga te the carboxylate functionality of D140, we further replaced D140 with glu tamic acid (D140E), phenylalanine (D140F), and asparagine (D140N). D140E de grades heme normally, but D140N shows reactivity similar to that of D140A. D140F loses heme degradation activity completely. All of these results indi cate that the carboxylate at position 140 is essential to activate the iron -bound dioxygen and hydroperoxide. On the basis of the present findings, we propose an oxygen activation mechanism involving the hydrogen-bonding netw ork through the bridging water and D140 side chain.