Role of the Triplet State and Protein Dynamics in the Formation and Stability of the Tryptophan Radical in an Apoazurin Mutant

Abstract

The protein, azurin, has enabled the study of the tryptophan radical. Upon UV excitation of tyrosine-deficient apoazurin and in the presence of a Co(III) electron acceptor, the neutral radical (W48•) is formed. The lifetime of W48• in apoazurin is 41 s, which is shorter than the lifetime of several hours in Zn-substituted azurin. Molecular dynamics simulations revealed enhanced fluctuations of apoazurin which likely destabilize W48•. The photophysics of W48 was investigated to probe the precursor state for ET. The phosphorescence intensity was eliminated in the presence of an electron acceptor while the fluorescence was unchanged; this quenching of the phosphorescence is attributed to ET. The kinetics associated with W48• were examined with a model that incorporates intersystem crossing, ET, deprotonation, and decay of the cation radical. The estimated rate constants for ET (6 × 10 6 s –1 ) and deprotonation (3 × 10 5 s –1 ) are in agreement with a photoinduced mechanism where W48• is derived from the triplet state. The triplet as the precursor state for ET was supported by photolysis of apoazurin with 280 nm in the absence and presence of triplet-absorbing 405 nm light. Absorption bands from the neutral radical were observed only in the presence of blue light.

Electrochemical and Structural Study of the Buried Tryptophan in Azurin: Effects of Hydration and Polarity on the Redox Potential of W48

“…Note that we observed no significant difference in protein stability between Cu- and Zn-forms of AzW48 (Table S4), though the apo-AzW48 is seen to exhibit a greatly reduced T m (Δ = −22.2 °C) and enthalpy (ΔΔ H ° = −160 kJ/mol) relative to those of the as-isolated form. The latter is consistent with recent MD simulations that predicted a destabilized protein structure for the metal-deficient azurin . Importantly, the peak potentials of Zn 2+ -substituted and as-isolated AzW48 samples are nearly identical, though the as-isolated sample produced slightly reduced currents .…”

“…The presence of water in the F110X mutants has implications for the PCET reaction for W48. Upon UV excitation, W48 undergoes ET followed by PT to generate the tryptophan neutral radical that has a decay time of 7 h. , The proton acceptor has not yet been identified, and given the lack of evidence of water in the hydrophobic W48 cavity, it has been hypothesized that the proton acceptor is the nearby F110 residue . However, protein fluctuations that permit transient interactions of W48 with water cannot be excluded .…”

“…Upon UV excitation, W48 undergoes ET followed by PT to generate the tryptophan neutral radical that has a decay time of 7 h. , The proton acceptor has not yet been identified, and given the lack of evidence of water in the hydrophobic W48 cavity, it has been hypothesized that the proton acceptor is the nearby F110 residue . However, protein fluctuations that permit transient interactions of W48 with water cannot be excluded . For the F110X mutants, the presence of an aqueous H-bond suggests that a water molecule in the W48 cavity could serve as the proton acceptor.…”

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

“…Long-lifetime, room temperature phosphorescence of azurin as well as other proteins has been reported . Several small molecules and proteins are known to quench tryptophan phosphorescence in azurin, − and ET has been suggested as a phosphorescence quenching mechanism. , A goal of this work is to demonstrate that 3 W48* is the parent ET state for formation of W48• in Zn-azurin (we recently reported similar results for apoazurin) . A Stern–Volmer analysis of phosphorescence quenching allows for the investigation of the quenching mechanisms with small molecule and protein quenchers; this analysis provides insights on protein–quencher interactions that affect the PCET reaction.…”

“…28,29 A goal of this work is to demonstrate that 3 W48* is the parent ET state for formation of W48• in Zn-azurin (we recently reported similar results for apoazurin). 30 A Stern−Volmer analysis of phosphorescence quenching allows for the investigation of the quenching mechanisms with small molecule and protein quenchers; this analysis provides insights on protein−quencher interactions that affect the PCET reaction.…”