Rejigging Electron and Proton Transfer to Transition between Dioxygenase, Monooxygenase, Peroxygenase, and Oxygen Reduction Activity: Insights from Bioinspired Constructs of Heme Enzymes

Abstract

Nature has employed heme proteins to execute a diverse set of vital life processes. Years of research have been devoted to understanding the factors which bias these heme enzymes, with all having a heme cofactor, toward distinct catalytic activity. Among them, axial ligation, distal super structure, and substrate binding pockets are few very vividly recognized ones. Detailed mechanistic investigation of these heme enzymes suggested that several of these enzymes, while functionally divergent, use similar intermediates. Furthermore, the formation and decay of these intermediates depend on proton and electron transfer processes in the enzyme active site. Over the past decade, work in this group, using in situ surface enhanced resonance Raman spectroscopy of synthetic and biosynthetic analogues of heme enzymes, a general idea of how proton and electron transfer rates relate to the lifetime of different O₂ derived intermediates has been developed. These findings suggest that the enzymatic activities of all these heme enzymes can be integrated into one general cycle which can be branched out to different catalytic pathways by regulating the lifetime and population of each of these intermediates. This regulation can further be achieved by tuning the electron and proton transfer steps. By strategically populating one of these intermediates during oxygen reduction, one can navigate through different catalytic processes to a desired direction by altering proton and electron transfer steps.

Figure 1

Active site structures of (A) cytochrome c (PDB ID: 1HRC), (B) cytochrome c oxidase (PDB ID: 1OCC), (C) myoglobin (PDB ID: 2 V1J), (D) cytochrome P450 (PDB ID: 1AKD), and (E) heme peroxidase (PDB ID: 2YLJ).

Figure 2

Oxidation of substrates by (A) peroxidase using H₂O₂ and (B) Cyt P450 using molecular O₂

Figure 3

Consolidated reactivity scheme for heme enzymes involved in O₂ activation. O₂ binding proteins (orange arrow) use O₂ to form Fe^IIIO₂^•– to carry and store O₂. Heme dioxygenase (purple arrow) uses the same intermediate to incorporate both the oxygen atoms of the molecular O₂ into organic substrates. CcO (black arrow) and Cyt P450 (golden arrow) enter into the cycle through the Fe^IIIO₂^•– intermediate but move forward in the cycle to form high valent intermediates and take part in the O₂ reduction and oxygenase mechanism, respectively. The prominent bifurcation of the reactivity happens after the formation of the high valent intermediate, and this branching is schematically represented in the figure using color coded arrows. On the other hand, heme peroxidase (red arrow) enters cycle using H₂O₂ by forming the Fe^III–OOH intermediate and then proceeds through the cycle to oxidize substrates. The formation of high valent intermediates with peroxides was also achieved using a similar technique for oxygen carrying enzymes and dioxygenase enzymes (dashed purple and orange arrows). Note that this does not always lead to any native enzymatic reaction.

Figure 4

Schematic representation of the branching pathways during the ORR by iron porphyrin systems. Here, L represents different axial ligands.

Figure 5

Pictorial representation of the (A) the mutually exclusive delivery nature of the protons and electrons in the Cyt P450 active site and (B) electrodes used for heterogeneous electrocatalytic purposes where ‘L’ depicts the axial ligands to the Iron porphyrin.

Figure 6

(A) Active site structure of CcO in an enzyme environment (1OCC) and (B) synthetic model of the CcO active site. (C) Computer model of G65YCu_BMb showing its catalytic center containing the distal Cu_B bound to three histidine and a tyrosine residue. Crystal structures of (D) V68ECuBMb and (E) I107E/V68ECuBMb showing the glutamate residues (yellow).

Figure 7

Schematic representation of the fabrication of the bioinspired electrodes.

Figure 8

(A) L⁶-FeCu and (B) Fe–Cu heme Cu oxidase model. Porphyrins with basic residues in the distal super structure: (C) FeL₂, (D) FeL₃, and (E) Fe-Marg.

Figure 9

(A) Active site of Cyt P450 (PDB ID: 1AKD) with thiolate bound heme. (B) Schematic representation of the competition between monooxygenation (blue arrow) and oxygen reduction (red arrow) by CytP450 family of enzymes.

Figure 10

Schematic representation of the monooxygenase activity depicting both the kinetic solvent isotope effect (KSIE) and kinetic isotope effect (KIE) during the oxygenation of C–H bond.

Figure 11

(A) Top view of iron-picket fence porphyrin (FePf) showing a substrate access cavity with a diameter of 6 Å. (B) The access of tertiary H of adamantane demands a conelike geometry resulting in steric hindrance. This accounts for the lesser selectivity toward tertiary C–H bond oxidation. (C) Secondary H accesses the cavity with a lesser amount of steric hindrance because of its trigonal architecture, resulting in more selectivity toward secondary C–H oxidation despite the higher BDE. (D) Wider substrate access cavity of iron-half picket fence (FehPf) depicting the lesser amount of bulky pivolyl groups in the secondary geometry.

Figure 12

(A) O₂ bound active site of heme dioxygenase (PDB ID: 5TI9). (B) Schematic representation of the formation of NFK by heme dioxygenase enzyme.

Figure 13

Schematic representation of (A) reaction of a pendant quinol molecule with a heme superoxide complex and (B) reaction of a synthetic heme superoxide adduct with an array of indole substrates in solution.

Figure 14

(A) Pictorial depiction of the electrochemical construct performing the dioxygenation of indole. (B) Schematic representation of the mechanism involved in the process of 4e- oxidation.

Figure 15

(A) Active site of heme peroxidase (PDB ID: 2CYP). (B) Interaction of the distal Arg and His with Fe^III–OOH.

Figure 16

Peroxidase activity of synthetic heme (A) Fe-MARG and (B) Fe-L₃.

Figure 17

(A) Tunneling lowers the observed barrier relative to the semi classical TS. (B) Schematic representation of the TS where L represents the different axial ligations.