New study shows why heme-copper oxidases prefer copper over iron

(Phys.org)—A family of enzymes known as heme-copper oxidases (HCOs) plays a pivotal role in the reduction of oxygen into water during cellular respiration. One mystery surrounding heme-copper oxidases is why the non-heme metal center tends to be copper rather than iron.

A group of researchers from the University of Illinois, Stevens Institute of Technology, and Oregon Health & Science University has developed a biosynthetic protein model system that replicates the active sites and many structural features of naturally-occurring HCOs. With their model system, they looked at the differences in the reduction of oxygen to water when the non-heme metal is iron versus when it is copper. They found that the non-heme metal plays a key role in electron donation and O-O bond cleavage and that it is likely copper's d-orbital electron configuration that causes its enhanced activity. Their work appears in Nature Chemistry.

"HCOs have been studied for more than half a decade now, but the selection of copper by nature at the nonheme center over other metal ions was not understood," says lead author Dr. Ambika Bhagi-Damodaran. "Our work is exciting because we finally resolve this long-standing question regarding the structure and function of this very important respiratory enzyme."

Their synthetic analog to the natural heme-copper oxidase is made from myoglobin, a small protein found in muscle that is a cousin to hemoglobin. Myoglobin has an iron-containing heme center and is denoted as Fe_BMb(Fe^II) because the heme iron is in the 2+ oxidation state. Fe_BMb(Fe^II) was produced and purified without a metal in the non-heme site using a previously reported procedure.

The empty Fe_BMb(Fe^II) was titrated with Zn^II, which is not redox active and serves as the experimental control, Cu^I, and Fe^II. Ultraviolet-visible spectroscopy confirmed that each metal was incorporated into the non-heme site. X-ray crystallography confirmed that each of these metal-Fe_BMb(Fe^II) variants exhibited similar active sites. In other words, this confirmed that the identity of the non-heme metal did not induce structural changes.

Bhagi-Damodaran et al. then investigated the differences in catalytic activity between the iron- and copper-containing species. They looked at reaction rate as well as product selectivity. Their enzymatic assay showed that Fe^II-Fe_BMb(Fe^II) and Cu^I-Fe_BMb(Fe^II) had 11-fold and 30-fold higher oxidase activity compared to the Zn^II control and the Fe_BMb(Fe^II) without a non-heme metal.

Using electron paramagnetic resonance and X-ray near-edge spectroscopy, they determined that the non-heme Cu^I was oxidized to Cu^II and the non-heme Fe^II was oxidized to Fe^II, thus confirming that the non-heme metal plays a central role in oxygen reduction as electron donors. To further understand the difference between copper and iron, Bhagi-Damodaran et al. studied the standard reduction potentials (E^o') of Fe^III/Fe^II and Cu^II/Cu^I at the model enzyme site. (The heme iron was replaced with a redox-inactive zinc protoporphyrin using a previously reported protocol.)

They found in both species a single reversible wave that corresponded to E^o' of 259 ± 20mV for iron and E^o' of 387 ± 25mV for copper. Since standard reduction potentials are related to the thermodynamic driving force of a reaction, copper's higher value means that Cu^II/Cu^I is more efficient at receiving electrons for the electrochemical reduction of oxygen to water.

The last step was to look at whether non-heme iron or copper interacts with heme-bound O₂ to aid in cleaving the O-O bond. Using resonance Raman spectroscopy, the authors looked at the vibration of the O-O bond and found that the terminal oxygen atom interacts with the nonheme metal weakening the O-O bond. To test if the identity of the nonheme metal had any impact on O-O bond length, the authors performed Density Functional Theory calculations and found that O-O bond length was longest in Cu^I-Fe_BMb(Fe^II). These results showed that the non-heme metal plays an important role in activating the oxygen molecule and facilitating O-O cleavage.

The preference of HCOs for copper is likely due to the higher redox potential of Cu as well as its d-orbital electron configuration. Copper-II has nine d-electrons, while Fe^III has five d-electrons. This additional electron density gives copper the advantage in orbital interactions with oxygen's highest occupied molecular orbitals.

Overall, this work provides important insights into naturally-occurring heme-copper complexes. According to corresponding author Professor Yi Lu, "We anticipate our work to be a starting point for more focused efforts toward using different metal ions at the non-heme site for various biochemical reactions. This pursuit can aid the design of novel catalysts required in alternative energy technologies and other biotechnological applications."

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