Density functional study for the bridged dinuclear center based on a high-resolution X-ray crystal structure of ba3 cytochrome c oxidase from Thermus thermophilus

Abstract

Strong electron density for a peroxide type dioxygen species bridging the Fea3 and CuB dinuclear center (DNC) was observed in the high-resolution (1.8 Å) X-ray crystal structures (PDB entries 3S8G and 3S8F) of ba3 cytochrome c oxidase (CcO) from Thermus thermophilus. The crystals represent the as-isolated X-ray photoreduced CcO structures. The bridging peroxide was proposed to arise from the recombination of two radiation-produced HO(•) radicals formed either very near to or even in the space between the two metals of the DNC. It is unclear whether this peroxide species is in the O2(2-), O2(•)(-), HO2(-), or the H2O2 form and what is the detailed electronic structure and binding geometry including the DNC. In order to answer what form of this dioxygen species was observed in the DNC of the 1.8 Å X-ray CcO crystal structure (3S8G), we have applied broken-symmetry density functional theory (BS-DFT) geometric and energetic calculations (using OLYP potential) on large DNC cluster models with different Fea3-CuB oxidation and spin states and with O2(2-), O2(•)(-), HO2(-), or H2O2 in the bridging position. By comparing the DFT optimized geometries with the X-ray crystal structure (3S8G), we propose that the bridging peroxide is HO2(-). The X-ray crystal structure is likely to represent the superposition of the Fea3(2+)-(HO2(-))-CuB(+) DNC's in different states (Fe(2+) in low spin (LS), intermediate spin (IS), or high spin (HS)) with the majority species having the proton of the HO2(-) residing on the oxygen atom (O1) which is closer to the Fea3(2+) site in the Fea3(2+)-(HO-O)(-)-CuB(+) conformation. Our calculations show that the side chain of Tyr237 is likely trapped in the deprotonated Tyr237(-) anion form in the 3S8G X-ray crystal structure.

Figure 1

The heme-a₃ and Cu_B dinuclear center (DNC) observed in the ba₃ CcO X-ray crystal structure 3S8G from Tt.

Figure 2

The water molecules and the H-bonding residue sidechains above the DNC.

Figure 3

The initial structure of the Fe³⁺-O₂²⁻-Cu²⁺, Fe²⁺-O₂^·−-Cu¹⁺, and Fe²⁺-O₂²⁻-Cu¹⁺ DNC model clusters. It is basically the combination of Fig. 1 and Fig. 2 with H_link atoms. The central Fe-O1-O2-Cu portion is also shown in Fig. 4 for the sake of clarity.

Figure 4

The detailed central Fe-O1-O2-Cu conformation of Fig. 3 for the Fe³⁺-O₂²⁻-Cu²⁺, Fe²⁺-O₂^·−-Cu¹⁺, and Fe²⁺-O₂²⁻-Cu¹⁺ DNC model clusters. The Fe²⁺-O₂^·−-Cu¹⁺ model has one more electron than the Fe³⁺-O₂²⁻-Cu²⁺ cluster, and one less electron than the Fe²⁺-O₂²⁻-Cu¹⁺ center.

Figure 5

The central DNC of the starting conformation of the Fe²⁺-(HO-O)⁻-Cu¹⁺ state for geometry optimization, where a proton binds with O1.

Figure 6

The central DNC of the starting conformation of the Fe²⁺-(OOH)⁻-Cu¹⁺ state, where a proton binds with O2 and H-bonds with HOH608.

Figure 7

The central DNC of the starting conformation of the cluster in Fe²⁺-HOOH-Cu¹⁺ state.

Figure 8

Comparing the positions of Fe, N(His384), O1, O2, Cu, N(His233), N(His282), and N(His283) in the DNC’s of 3S8G.pdb (blue) and Fe^2+,LS-O₂^·−-Cu¹⁺ (red). The RMSD between these two 8-atom structures is 0.46 Å (see Table 2)

Figure 9

The central portion of the DNC of the optimized Fe^2+,LS-(OOH)⁻-Cu¹⁺ cluster, where a proton binds with O2.

Figure 10

The central portion of the DNC of the optimized Fe^2+,IS-(OOH)⁻-Cu¹⁺ cluster, where a proton binds with O2. The optimized Fe^2+,HS-(O-OH)⁻-Cu¹⁺ structure is very similar to this.

Figure 11

The central portion of the DNC of the optimized Fe^2+,LS-(HO-O)⁻-Cu¹⁺ cluster, where a proton binds with O1.

Figure 12

The central portion of the DNC of the optimized Fe^2+,IS-(HO-O)⁻-Cu¹⁺ cluster, where a proton binds with O1. The optimized Fe^2+,HS-(HO-O)⁻-Cu¹⁺ structure is very similar to this.

Figure 13

The overlap of the DNC of the 3S8G X-ray crystal structure (in blue, without water molecules) and the Average-xyz (in red) structure of the three Fe^2+,LS-(HO-O)⁻-Cu¹⁺(Y237⁻), Fe^2+,IS-(HO-O)⁻-Cu¹⁺(Y237⁻) and Fe^2+,HS-(HO-O)⁻-Cu¹⁺(Y237⁻) optimized clusters.

Figure 14

Comparing the positions of Fe, N(His384), O1(H), O2, Cu, N(His233), N(His282), and N(His283) in the DNC’s of 3S8G.pdb (blue) and Fe^2+,LS-(HO-O)⁻-Cu¹⁺(Y237⁻)⁽³⁾ (red).