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The structure of plastocyanin tunes the midpoint potential by restricting axial ligation of the reduced copper ion.

ABSTRACT: Blue copper proteins are models for illustrating how proteins tune metal properties. Nevertheless, the mechanisms by which the protein controls the metal site remain to be fully elucidated. A hindrance is that the closed shell Cu(I) site is inaccessible to most spectroscopic analyses. Carbon deuterium (C-D) bonds used as vibrational probes afford nonperturbative, selective characterization of the key cysteine and methionine copper ligands in both redox states. The structural integrity of Nostoc plastocyanin was perturbed by disrupting potential hydrogen bonds between loops of the cupredoxin fold via mutagenesis (S9A, N33A, N34A), variably raising the midpoint potential. The C-D vibrations show little change to suggest substantial alteration to the Cu(II) coordination in the oxidized state or in the Cu(I) interaction with the cysteine ligand. They rather indicate, along with visible and NMR spectroscopy, that the methionine ligand distinctly interacts more strongly with the Cu(I) ion, in line with the increases in midpoint potential. Here we show that the protein structure determines the redox properties by restricting the interaction between the methionine ligand and Cu(I) in the reduced state.

SUBMITTER: Mammoser CC

PROVIDER: S-EPMC10447441 | biostudies-literature | 2023 Aug

REPOSITORIES: biostudies-literature

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The structure of plastocyanin tunes the midpoint potential by restricting axial ligation of the reduced copper ion.

Mammoser Claire C CC LeMasters Brynn E BE Edwards Sydney G SG McRae Emma M EM Mullins M Hunter MH Wang Yiqi Y Garcia Nicholas M NM Edmonds Katherine A KA Giedroc David P DP Thielges Megan C MC

Communications chemistry 20230823 1

Blue copper proteins are models for illustrating how proteins tune metal properties. Nevertheless, the mechanisms by which the protein controls the metal site remain to be fully elucidated. A hindrance is that the closed shell Cu(I) site is inaccessible to most spectroscopic analyses. Carbon deuterium (C-D) bonds used as vibrational probes afford nonperturbative, selective characterization of the key cysteine and methionine copper ligands in both redox states. The structural integrity of Nostoc ...[more]

PMID: 37612467

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Project description:Interactions of the axial ligand with its blue copper center are known to be important in tuning spectroscopic and redox properties of cupredoxins. While conversion of the blue copper center with a weak axial ligand to a green copper center containing a medium strength axial ligand has been demonstrated in cupredoxins, converting the blue copper center to a red copper center with a strong axial ligand has not been reported. Here we show that replacing Met121 in azurin from Pseudomonas aeruginosa with Cys caused an increased ratio (R(L)) of absorption at 447 nm over that at 621 nm. Whereas no axial Cu-S(Cys121) interaction in Met121Cys was detectable by extended X-ray absorption fine structure (EXAFS) spectroscopy at pH 5, similar to what was observed in native azurin with Met121 as the axial ligand, the Cu-S(Cys121) interaction at 2.74 A is clearly visible at higher pH. Despite the higher R(L) and stronger axial Cys121 interaction with Cu(II) ion, the Met121Cys variant remains largely a type 1 copper protein at low pH (with hyperfine coupling constant A( parallel) = 54 x 10(-4) cm(-1) at pH 4 and 5), or distorted type 1 or green copper protein at high pH (A(parallel) = 87 x 10(-4) cm(-1) at pH 8 and 9), attributable to the relatively long distance between the axial ligand and copper and the constraint placed by the protein scaffold. To shorten the distance between axial ligand and copper, we replaced Met121 with a nonproteinogenic amino acid homocysteine that contains an extra methylene group, resulting in a variant whose spectra (R(L)= 1.5, and A(parallel) = 180 x 10(-4) cm(-1)) and Cu-S(Cys) distance (2.22 A) are very similar to those of the red copper protein nitrosocyanin. Replacing Met121 with Cys or homocysteine resulted in lowering of the reduction potential from 222 mV in the native azurin to 95 +/- 3 mV for Met121Cys azurin and 113 +/- 6 mV for Met121Hcy azurin at pH 7. The results strongly support the "coupled distortion" model that helps explain axial ligand tuning of spectroscopic properties in cupredoxins, and demonstrate the power of using unnatural amino acids to address critical chemical biological questions.

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The structure of plastocyanin tunes the midpoint potential by restricting axial ligation of the reduced copper ion.

Publications

The structure of plastocyanin tunes the midpoint potential by restricting axial ligation of the reduced copper ion.

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