In many reduced blue copper proteins the C-terminal surface-exposed active-site histidine protonates at low pH and dissociates from the Cu atom. In this state, the proteins exhibit high reduction potentials and low oxidation rates. In contrast, the homologous histidine (117) of azurin does not protonate. This difference has been examined by studying the electrochemical behavior of an azurin mutant in which histidine 117 is replaced by glycine to create a cavity enabling external ligands to enter the protein and coordinate to the Cu. We show that the external ligands influence the electrochemical properties of the copper site, as studied with potentiometric titrations of protein solutions and with fast-scan and low-temperature cyclic voltammetry of protein films adsorbed on graphite electrodes. The reduction potential (E 0′ ) of His117Gly azurin without external ligands is very high, at 670 ( 10 mV, but it decreases upon addition of Clor imidazole. The reduced form has little affinity for these ligands; however, under fast-scan or cryoscopic conditions (-70 °C, 70% methanol) the reduced form of the imidazole complex can be "trapped", and a reversible redox couple is established. The electrochemical kinetics of the trapped state are very fast and similar to those of wild-type (wt) azurin. The reduction potential is ∼60 mV lower than for wt azurin under identical conditions. The dissociation constant K diss of the Cu(I)-imidazole complex lies between 14 and 69 M at 20 °C, while that of the Cu(I)-Clcomplex is estimated to be as high as 10 6 M. These very low affinities show that for wt azurin the covalent link between the imidazole side chain of His117 and the protein framework is crucial for maintaining this side chain as a ligand of the Cu(I) ion.
The effects of different Cl- depletion treatments in photosystem II (PS-II)-enriched membranes have been investigated by electron paramagnetic resonance (EPR) spectroscopy and by measurements of oxygen-evolving activity. The results indicated that the oxygen-evolving complex of PS-II exhibits two distinct Cl(-)-dependent properties. (1) After Cl(-)-free washes at pH 6.3, a reversibly altered distribution of structural states of PS-II was observed, manifested as the appearance of a g = 4 EPR signal from the S2 state in a significant fraction of centers (20-40%) at the expense of the S2 multiline signal. In addition, small but significant changes in the shape of the S2 multiline EPR signal were observed. Reconstitution of Cl- to Cl(-)-free washed PS-II rapidly reversed the observed effects of the Cl(-)-free washing. The anions, SO4(2-) and F-, which are often used during Cl- depletion treatments, had no effect on the S2 EPR properties of PS-II under these conditions in the absence or presence of Cl-. Flash experiments and measurements of oxygen evolution versus light intensity indicated that the two structural states observed after the removal of Cl- at pH 6.3 originated from oxygen-evolving centers exhibiting a lowered quantum yield of water oxidation. (2) Depletion of Cl- in PS-II by pH 10 treatment reversibly inhibited the oxygen-evolving activity to approximately 15%. The pH 10 treatment depleted the Cl- from a site which is considered to be equivalent to that studied in most earlier work on Cl(-)-depleted PS-II. The S2 state in pH 10/Cl(-)-depleted PS-II was reversibly modified to a state from which no S2 multiline EPR signal was generated and which exhibited an intense S2 g = 4 EPR signal corresponding to at least 40% of the centers but possibly to a much larger fraction of centers. The state responsible for the intense S2 g = 4 signal generated under these conditions is unlike that observed after removal of Cl- from PS-II at pH 6.3, in that this state was more stable in the dark, showing a half-decay time of approximately 1.5 h at 0 degrees C, and was unable to undergo further charge accumulation. Nevertheless, a fraction of centers, probably different from those exhibiting the S2 g = 4 signal, was able to advance to the formal S3 state, giving rise to a narrow EPR signal around g = 2. Addition of the anions SO4(2-) or F- to pH 10/Cl(-)-depleted PS-II affected the properties of PS-II, resulting in EPR properties of the S2 state similar to those reported earlier following Cl- depletion treatment of PS-II in the presence of these anions. Surprisingly, after addition of F-, the g = 4 EPR signal showed a damped flash-dependent oscillation. In addition, a narrow signal around g = 2, corresponding to the formal S3 state, also showed a damped flash-dependent oscillation pattern. The presence of oscillating EPR signals (albeit damped) in F(-)-treated pH 10/Cl(-)-depleted PS-II indicates functional enzyme turnover. This was confirmed by measurements of the oxygen-evolving activity versus light intensity which indicat...
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.