Role of Heme Types in Heme-Copper Oxidases:  Effects of Replacing a Hemebwith a HemeoMimic in an Engineered Heme-Copper Center in Myoglobin^§

Abstract

To address the role of the secondary hydroxyl group of heme a/o in heme-copper oxidases, we incorporated Fe(III)-2,4 (4,2) hydroxyethyl vinyl deuterioporphyrin IX, as a heme o mimic, into the engineered heme-copper center in myoglobin (sperm whale myoglobin L29H/F43H, called Cu(B)Mb). The only difference between the heme b of myoglobin and the heme o mimic is the substitution of one of the vinyl side chains of the former with a hydroxyethyl group of the latter. This substitution resulted in an approximately 4 nm blue shift in the Soret band and approximately 20 mV decrease in the heme reduction potential. In a control experiment, the heme b in Cu(B)Mb was also replaced with a mesoheme, which resulted in an approximately 13 nm blue shift and approximately 30 mV decrease in the heme reduction potential. Kinetic studies of the heme o mimic-substituted Cu(B)Mb showed significantly different reactivity toward copper-dependent oxygen reduction from that of the b-type Cu(B)Mb. In reaction with O2, Cu(B)Mb with a native heme b showed heme oxygenase activity by generating verdoheme in the presence of Cu(I). This heme degradation reaction was slowed by approximately 19-fold in the heme o mimic-substituted Cu(B)Mb (from 0.028 s(-1) to 0.0015 s(-1)), while the mesoheme-substituted Cu(B)Mb shared a similar heme degradation rate with that of Cu(B)Mb (0.023 s(-1)). No correlation was found between the heme reduction potential and its O2 reactivity. These results strongly suggest the critical role of the hydroxyl group of heme o in modulating heme-copper oxidase activity through participation in an extra hydrogen-bonding network.

The Second Step of the Nitric Oxide Synthase Reaction: Evidence for Ferric-Peroxo as the Active Oxidant

“…[23][24][25] Increased electron density on the metal center should increase the rate of heterolysis to Cpd I by enhancing the "push effect," 26 while also stabilizing the resulting Cpd I species. With peroxidases, meso-heme substitution leads to increases in peroxide cleavage.…”

Role of Copper Ion in Regulating Ligand Binding in a Myoglobin-Based Cytochrome c Oxidase Model

“…27,28 Biochemical studies show that Cu B Mb binds copper tightly, with an equilibrium dissociation constant ( K d ) of 9 μ M. 27 In addition, upon exposure to atmospheric oxygen, Cu B Mb forms a stable O 2 adduct, albeit with a lower affinity as compared to the wild-type protein 27,28 Addition of Cu + to Cu B Mb facilitates O 2 binding and reduction (by receiving an electron from Cu + like that found in C c O); however, instead of producing the ferryl-heme intermediate as observed in the C c O reaction, it promotes the degradation of the prosthetic heme to verdoheme, 28 presumably a result of the lack of protons required for heterolytic cleavage of the O-O bond. 29 In addition to the oxygen reaction, Cu B Mb also exhibits NO reductase activity 30 in the presence of Cu + , but not in its absence, with an efficiency similar to that of TtC c O, 9 manifesting the important role of the copper ion in the catalytic properties of Cu B Mb.…”

Design and engineering of artificial oxygen-activating metalloenzymes

“…The rate with Mn· 4 ·apo-H64D/A71GMb was found to be threefold higher than that with Mn· 2 ·apo-H64D/A71GMb, thus confirming that the enlargement of the substrate cavity increase the accessibility of the substrate. 233 Substitutions at the 3- 3’- positions strongly affect the enantioselectivity of the sulfoxidation reaction. Interestingly, while Mn·4·apo-H64D/A71GMb showed 32% ee ( R ) selectivity, introduction of bulkier groups at the 3,3’-positions induces relative S -selectivity to end up 13% ( S ) for 6 .…”