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We first expressed and purified FeBMb without any nonheme metal at the FeB site

(henceforth referred to as the empty-FeBMb or simply E-FeBMb) into which any of the

desired nonheme metal (either Fe(II), Cu(I) or Zn(II)) could be selectively incorporated to yield the three different FeBMb variants (Fe(II)-FeBMb, Cu(I)-FeBMb and Zn(II)-FeBMb,

respectively) (see SI for more information on the protocol of preparation). The incorporation of a nonheme metal close to the heme center is easily discernable in UV- vis studies (Fig. 3.2D) as a shift in the heme Soret (bathochromic shift from 433 nm to 434 nm) and a peak splitting in visible absorption regions (the 557 nm peak of E-FeBMb

splits into two shallow bands at 550 nm and 572 nm after nonheme metal incorporation). A controlled study of the role of the nonheme metal is possible only if difference between the various FeBMb variants is the identity of the nonheme metal ion itself, while all other

structural features including the nonheme metal coordination sphere and metal-metal distances remain unchanged. To confirm that this is indeed the case, we have compared the crystal structures for the synthesized FeBMb variants (Fig. 3.2A-C), which show that,

for all three variants, the nonheme metals are coordinated to three histidines (H64, H43, H29) and a glutamate (68E) in a distorted trigonal-bipyramidal geometry, thereby conserving the desired coordination sphere. Furthermore, the heme-nonheme metal distances remain the same (~4.5 Å) within the experimental error of the x-ray crystallography resolution (~1.7 Å). In all, these studies confirm that the FeBMb variants

constitute a model system to probe different nonheme metals within the same protein scaffold, and holds potential to elucidate the preference for copper exhibited by native HCOs.

Armed with such a model system of FeBMb variants, we proceed to directly probe

the role of nonheme metal on oxidase activity (Fig. 3.2E). This includes the overall oxygen reduction rates (k), as well as the product selectivity (S) – an indicator of the quality of the oxygen reduction reaction. S is defined as the ratio of water produced (the desired end product and a consequence of a 4e- _{oxygen reduction process) to products of incomplete}

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(collectively known as reactive oxygen species or ROS). From a biological perspective, the formation of ROS not only decreases HCO efficiency, but also damages cellular biomolecules. In this study, we measured the overall oxygen reduction rates for various FeBMb variants under multi-turnover conditions using an O2 electrode (with N,N,N',N'-

tetramethyl-p-phenylenediamine (TMPD) as a redox mediator, and ascorbate as a

Figure 3.2. Crystal structure of Fe_BMb mutant (I107E- Fe_BMb) showing the catalytic heme- nonheme heterobinuclear metal center with Zn(II) (A, PDB: 3M3B), Fe(II) (B, PDB: 3M39) and Cu(II) (C, PDB: 3M3A) nonheme metal ions bound. The heme and amino acid residues as shown in licorice while the nonheme metal ions are shown in VDW representation as a sphere. (D) The UV-Vis spectroscopic measurements on Fe_BMb variants displaying the changes in the Soret (420-445 nm) and visible (500-700 nm) region on incorporation of nonheme Fe(II) and Cu(I) metal ions. (E) The rates of O₂ reduction to form either H₂O (blue) or ROS (red) catalyzed by 18 M Fe_BMb variants in 100 mM phosphate buffer (pH 6) containing ~250 M O₂, 1.8 mM TMPD, and 18 mM ascorbate.

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reductant) and a protocol reported previously for both native HCOs and their models.16,19

A slightly modified approach employing catalase16 _{was used to determine the product}

selectivity in these reactions. We also included E-FeBMb in addition to the three FeBMb

variants as a control. The oxygen reduction reaction with E-FeBMb (18 μM) showed an

initial rapid decrease in O2 concentration (by an amount corresponding to ~1 equivalent

of protein concentration) from the binding of O2 to the heme. This was followed by a slow

consumption of O2 with an initial rate of 0.21 (± 0.03) M/s of which only ~59% of O2 was

converted to water and rest to ROS (Fig. 3.3A). The oxygen reduction rates and product selectivity for the Zn(II)-FeBMb (k=0.22(± 0.02), S = 57%) are similar to those of E-FeBMb.

In contrast, both Fe(II)- and Cu(I)- substituted FeBMb variants showed dramatic

enhancements in oxygen reduction rates and product selectivity (k= 1.15 ± 0.07 M/s and

S = 96% for Fe, and k = 2.72 ± 0.1/s and S=94% for Cu). More specifically, the oxidase

activity (defined as the rate of water production) of Fe(II)-FeBMb and Cu(I)-FeBMb were

11-fold and 30-fold higher, respectively, than that of Zn(II)-FeBMb. We also note that

controls with wild-type myoglobin (WTMb, without the engineered FeB center) and in the

presence of Zn(II), Fe(II) and Cu(I) ions showed similar water production rates as E- FeBMb, but with a high excess of undesirable ROS production (Fig. 3.3B)35, further

Figure 3.3. (A) The results of the oxygen reduction enzymatic assay performed on the

oxygen electrode. The experiments were performed on 18 M FeBMb variants in 100 mM

phosphate buffer (pH 6) containing 250 M oxygen, 1.8 mM TMPD, and 18 mM ascorbate. (B) The rates of oxygen reduction to form either water (blue) or ROS (red) catalyzed by 18 M FeBMb variants in 100 mM phosphate buffer (pH 6) containing 250 M oxygen, 1.8 mM

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confirming that enhancements in oxidase activity occur only when Fe(II) and Cu(I) are coordinated to the nonheme FeB center.

These experiments beget several important conclusions. To begin with, they confirm that the synthesized Fe(II)- and Cu(I)- FeBMb variants are excellent functional

mimics of native HCO’s. Second, the fact that the oxidase activity of redox-inactive Zn(II)- substituted variant remains the same as E-FeBMb as compared to dramatic

enhancements seen in its redox active analogues (Fe(II)- and Cu(I)) establishes, for the first time, the unequivocal role of the non-heme metal as a an electron donor in the oxygen reduction. In addition, these experiments also demonstrate the superior performance of Cu over Fe as a nonheme metal for oxygen reduction reactions, and suggest a functional reason for Nature’s choice of Cu in HCO’s. We can proceed to understand the chemical origin for the superior performance of Cu over Fe by considering all differences that can arise from the identity of the non-heme metal. These include: 1) an increase in the redox potential (E°ˊ) of either the heme or nonheme metal ion that can enhance electron transfer rates at the catalytic center and 2) changes to the O-O bond length that can arise from differences in the electronic configuration of the nonheme metal. In the sections that follow, we explore in detail each of these possibilities using a combination of electrochemical, kinetic and computational approaches.