Photoinduced Electron Transfer from the Tryptophan Triplet State in Zn-Azurin

Abstract

Tryptophan is one of few residues that participates in biological electron transfer reactions. Upon substitution of the native Cu²⁺ center with Zn²⁺ in the blue-copper protein azurin, a long-lived tryptophan neutral radical can be photogenerated. We report the following quantum yield values for Zn-substituted azurin in the presence of the electron acceptor Cu(II)-azurin: formation of the tryptophan neutral radical (Φ_rad), electron transfer (Φ_ET), fluorescence (Φ_fluo), and phosphorescence (Φ_phos), as well as the efficiency of proton transfer of the cation radical (Φ_PT). Increasing the concentration of the electron acceptor increased Φ_rad and Φ_ET values and decreased Φ_phos without affecting Φ_fluo. At all concentrations of the acceptor, the value of Φ_PT was nearly unity. These observations indicate that the phosphorescent triplet state is the parent state of electron transfer and that nearly all electron transfer events lead to proton loss. Similar results regarding the parent state were obtained with a different electron acceptor, [Co(NH₃)₅Cl]²⁺; however, Stern-Volmer graphs revealed that [Co(NH₃)₅Cl]²⁺ was a more effective phosphorescence quencher (K _SV = 230 000 M^-1) compared to Cu(II)-azurin (K _SV = 88 000 M^-1). Competition experiments in the presence of both [Co(NH₃)₅Cl]²⁺ and Cu(II)-azurin suggested that [Co(NH₃)₅Cl]²⁺ is the preferred electron acceptor. Implications of these results in terms of quenching mechanisms are discussed.

Figure 1

Absorption (red) and normalized emission (blue) spectra of deoxygenated ZnAzW48 in 20 mM KPi, pH = 7.0. The normalized emission spectrum of NATA in air is shown as the green dashed curve. The inset shows the phosphorescence region of deoxygenated ZnAzW48 (solid) and ZnAzW48 in air (dashed).

Figure 2

Normalized emission spectra of 25 ± 2 μM ZnAzW48 with increasing concentrations of electron acceptor CuAzW48 (top) and 26 ± 1 μM ZnAzW48 with increasing concentrations of [Co(NH₃)₅Cl]²⁺ (bottom). The ratios [CuAz]/[ZnAz] for these representative trials were 0, 0.26, 0.51, 0.80, 1.1, 1.5, 2.0, and 3.2, and [Co(III)]/[ZnAz] were 0, 0.10, 0.17, 0.24, 0.46, and 1.9. Insets show the expanded region of the phosphorescence.

Figure 3

Stern–Volmer plot of ZnAzW48 showing the relative phosphorescence (Φ_phos⁰/Φ_phos, open markers) and relative fluorescence (Φ_fluo⁰/Φ_fluo, filled markers) quantum yields with acceptors CuAzW48 (blue squares) and [Co(NH₃)₅Cl]²⁺ (red circles) at T = 22 °C. The phosphorescence data were fit to the linear Stern–Volmer equation (solid line), and the resulting values of K_SV (slope, units M^–1) and y-intercepts are indicated. Data are shown up to [CuAz]/[ZnAz] =1.5 and [Co(III)]/[ZnAz] = 0.46.

Figure 4

Top panel: Representative absorption spectra during the photolysis of ZnAzW48 in the presence of CuAzW48 with [CuAz]/[ZnAz] = 0.80 for 1 to 40 min. The black curve is the prephotolysis spectrum. Inset shows the region of the isosbestic point. Bottom panel: Difference spectra calculated by subtracting the prephotolysis spectrum from each spectrum on the top panel. The difference spectra have been corrected for the 628 nm bleach. The inset shows the increase in concentration of W48• in ZnAzW48 and decrease in concentration (bleach) of the Cu(II) metal center in CuAzW48.

Figure 5

Representative difference spectra for the photolysis of ZnAzW48 in the presence of [Co(NH₃)₅Cl]²⁺ with [Co(III)]/[ZnAz] = 1.9 for 3 to 110 min. The inset shows the increase in concentration of W48•.

Figure 6

Growth of the 515 nm absorbance (top, open symbols) and decay of the 628 nm absorbance (bottom, filled symbols) during photolysis of ZnAzW48 in the presence of CuAzW48 or both [Co(NH₃)₅Cl]²⁺ and CuAzW48 electron acceptors. The concentrations are as follows: Black squares [ZnAzW48] = 23 μM and [CuAzW48] = 29 μM; green circles [ZnAzW48] = 24 μM, [CuAzW48] = 29 μM, and [Co(NH₃)₅Cl]²⁺ = 4 μM; red triangles [ZnAzW48] = 26 μM, [CuAzW48] = 31 μM, and [Co(NH₃)₅Cl]²⁺ = 7 μM; blue stars [ZnAzW48] = 28 μM, [CuAzW48] = 35 μM, and [Co(NH₃)₅Cl]²⁺ = 34 μM.

Figure 7

Left axis: Relative quantum yields for fluorescence (Φ_fluo/Φ_fluo⁰, black filled circle) and phosphorescence (Φ_phos/Φ_phos⁰, black open circle) for ZnAzW48 in the presence of CuAzW48 (top panel) and [Co(NH₃)₅Cl]²⁺ (bottom panel). Right axis (blue and red curves): Quantum yields for radical formation (Φ_rad, blue filled circle) and ET (Φ_ET, red filled circle). Lines connect the points to help guide the eye.

Figure 8

Relevant relaxation pathways and measured quantum yields for photoexcited W48 in ZnAzW48 in the presence (right path) and absence (left path) of electron acceptor; all values are from Table 1. In the presence of the CuAzW48 electron acceptor [CuAz]/[ZnAz] = 2.4 ± 0.5), the triplet state relaxes via ET to the Cu(II) metal center and the value of Φ_phos is negligible at 0.003. In the absence of an electron acceptor, ET from T₁ is not possible and Φ_phos is high at 0.018. In contrast to Φ_phos, the value of Φ_fluo is not significantly affected by the presence of an electron acceptor.