Axial interactions in the mixed-valent Cu _A active site and role of the axial methionine in electron transfer

Abstract

Within Cu-containing electron transfer active sites, the role of the axial ligand in type 1 sites is well defined, yet its role in the binuclear mixed-valent Cu A sites is less clear. Recently, the mutation of the axial Met to Leu in a Cu A site engineered into azurin (Cu A Az) was found to have a limited effect on E 0 relative to this mutation in blue copper (BC). Detailed low-temperature absorption and magnetic circular dichroism, resonance Raman, and electron paramagnetic resonance studies on Cu A Az (WT) and its M123X (X = Q, L, H) axial ligand variants indicated stronger axial ligation in M123L/H. Spectroscopically validated density functional theory calculations show that the smaller ΔE 0 is attributed to H 2 O coordination to the Cu center in the M123L mutant in Cu A but not in the equivalent BC variant. The comparable stabilization energy of the oxidized over the reduced state in Cu A and BC (Cu A ∼ 180 mV; BC ∼ 250 mV) indicates that the S(Met) influences E 0 similarly in both. Electron delocalization over two Cu centers in Cu A was found to minimize the Jahn-Teller distortion induced by the axial Met ligand and lower the inner-sphere reorganization energy. The Cu-S(Met) bond in oxidized Cu A is weak (5.2 kcal/ mol) but energetically similar to that of BC, which demonstrates that the protein matrix also serves an entatic role in keeping the Met bound to the active site to tune down E 0 while maintaining a low reorganization energy required for rapid electron transfer under physiological conditions. spectroscopy | reduction potential | energy transduction pathway L ong-range electron transfer (ET) is vital to a wide range of biological processes, including two key energy transduction pathways essential for life: H 2 O oxidation in photosynthesis and O 2 reduction in respiration (1, 2). Nature has adapted a conserved cupredoxin fold motif (i.e., the Greek-key β barrel) to construct two evolutionarily linked, but structurally distinct Cucontaining ET proteins (3-5). These are the mononuclear type 1 (T1) or blue copper (BC) and binuclear purple Cu A proteins. The first coordination sphere of the classic BC sites [e.g., plastocyanin (Pc) and azurin (Az)] consists of a trigonally distorted tetrahedral environment where Cu resides in an equatorial plane formed by one S(Cys) and two N(His) ligands and has an axial S (Met) ligand (Fig. 1A) (Fig. 1B) (8-11). Both sites carry out rapid, efficient longrange ET with rates on the order of 10 3 -10 5 s −1 (12, 13).Although BC proteins use a Cu + /Cu 2+ redox couple, the binuclear Cu A sites use a (Cu 1+ -Cu 1+ )/(Cu 1.5+ -Cu 1.5+ ) redox cycle. The oxidized form of Cu A is mixed-valent (MV), with a highly covalent Cu 2 S 2 core that gives rise to its unique spectroscopic features. The unpaired electron is fully delocalized over the two Cu centers and exhibits a characteristic seven-line 63,65 Cu hyperfine splitting pattern in electron paramagnetic resonance (EPR) spectroscopy (14, 15). Maintaining valence delocalization even in the presence of a low symmetry protein ...

Probing the role of the backbone carbonyl interaction with the CuA center in azurin by replacing the peptide bond with an ester linkage

“…axial position is consistent with previous reports of mutations at the other axial Met123 position 24,42 and leads us to conclude that the diamond core structure of Cu 2 S Cys2 shown in Figure 1B is resistant to variations of axial interactions.…”

“…Previous studies of Cu A azurin has shown that mutations of the axial Met to Glu, Gln or Leu resulted in only ∼ 20 mV changes of the reduction potentials of the Cu A azurin. 24 The lack of change in reduction potential observed for the amide to ester carbonyl change in another axial position is consistent with previous reports of mutations at the other axial Met123 position 24,42 and leads us to conclude that the diamond core structure of Cu 2 S Cys2 shown in Figure 1B is resistant to variations of axial interactions. In summary, a Cu A azurin variant consisting of an amide-to-ester replacement in the backbone has been prepared to investigate the properties of the carbonyl interaction with the Cu A center.…”

Metalloprotein entatic control of ligand-metal bonds quantified by ultrafast x-ray spectroscopy

“…While functioning as an ET protein, the Fe–S(Met) bond keeps the metal low-spin while the heme iron site shuttles between Fe(II) and Fe(III), which maintains a low inner-sphere reorganization energy for rapid ET (3, 4, 6). However, cyt c can also function as a peroxidase, which requires loss of the Fe(III)–S(Met) bond to open a ligand binding site for catalysis (7, 8).…”

Activating Metal Sites for Biological Electron Transfer

“…Parallel studies were done on the binuclear Cu A center in Fig 1, which defined an equivalent role of the protein stabilized thioether-Cu bond: to stabilize the oxidized over the reduced state, and to tune down the Cu A reduction potential. [25] From Fig 1, cytochrome c has a thioether-metal bond that was thought to play the opposite role in function. This is considered in the next section.…”