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Why Do Most Aromatics Fail to Support Hole Hopping in the Cytochrome c Peroxidase-Cytochrome c Complex?

ABSTRACT: Electron transport through aromatic species (especially tryptophan and tyrosine) plays a central role in water splitting, redox signaling, oxidative damage protection, and bioenergetics. The cytochrome c peroxidase (CcP)-cytochrome c (Cc) complex (CcP:Cc) is used widely to study interprotein electron transfer (ET) mechanisms. Tryptophan 191 (Trp191) of CcP supports hole hopping charge recombination in the CcP:Cc complex. Experimental studies find that when Trp191 is substituted by tyrosine, phenylalanine, or redox-active aniline derivatives bound in the W191G cavity, enzymatic activity and charge recombination rates both decrease. Theoretical analysis of these CcP:Cc complexes finds that the ET kinetics depend strongly on the chemistry of the modified Trp site. The computed electronic couplings in the W191F and W191G species are orders of magnitude smaller than in the native protein, due largely to the absence of a hopping intermediate and the large tunneling distance. Small molecules bound in the W191G cavity are weakly coupled electronically to the Cc heme, and the structural disorder of the guest molecule in the binding pocket may contribute further to the lack of enzymatic activity. The couplings in W191Y are not substantially weakened compared to the native species, but the redox potential difference for tyrosine vs tryptophan oxidation accounts for the slower rate in the Tyr mutant. Thus, theoretical analysis explains why only the native Trp supports rapid hole hopping in the CcP:Cc complex. Favorable free energies and electronic couplings are essential for establishing an efficient hole hopping relay in this protein-protein complex.

SUBMITTER: Ru X

PROVIDER: S-EPMC8466190 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:We have investigated photoinduced hole hopping in a Pseudomonas aeruginosa azurin mutant Re126WWCuI, where two adjacent tryptophan residues (W124 and W122) are inserted between the CuI center and a Re photosensitizer coordinated to a H126 imidazole (Re = ReI(H126)(CO)3(dmp)+, dmp = 4,7-dimethyl-1,10-phenanthroline). Optical excitation of this mutant in aqueous media (≤40 μM) triggers 70 ns electron transport over 23 Å, yielding a long-lived (120 μs) ReI(H126)(CO)3(dmp•-)WWCuII product. The Re126FWCuI mutant (F124, W122) is not redox-active under these conditions. Upon increasing the concentration to 0.2-2 mM, {Re126WWCuI}2 and {Re126FWCuI}2 are formed with the dmp ligand of the Re photooxidant of one molecule in close contact (3.8 Å) with the W122' indole on the neighboring chain. In addition, {Re126WWCuI}2 contains an interfacial tryptophan quadruplex of four indoles (3.3-3.7 Å apart). In both mutants, dimerization opens an intermolecular W122' → //*Re ET channel (// denotes the protein interface, *Re is the optically excited sensitizer). Excited-state relaxation and ET occur together in two steps (time constants of ∼600 ps and ∼8 ns) that lead to a charge-separated state containing a Re(H126)(CO)3(dmp•-)//(W122•+)' unit; then (CuI)' is oxidized intramolecularly (60-90 ns) by (W122•+)', forming ReI(H126)(CO)3(dmp•-)WWCuI//(CuII)'. The photocycle is closed by ∼1.6 μs ReI(H126)(CO)3(dmp•-) → //(CuII)' back ET that occurs over 12 Å, in contrast to the 23 Å, 120 μs step in Re126WWCuI. Importantly, dimerization makes Re126FWCuI photoreactive and, as in the case of {Re126WWCuI}2, channels the photoproduced "hole" to the molecule that was not initially photoexcited, thereby shortening the lifetime of ReI(H126)(CO)3(dmp•-)//CuII. Although two adjacent W124 and W122 indoles dramatically enhance CuI → *Re intramolecular multistep ET, the tryptophan quadruplex in {Re126WWCuI}2 does not accelerate intermolecular electron transport; instead, it acts as a hole storage and crossover unit between inter- and intramolecular ET pathways. Irradiation of {Re126WWCuII}2 or {Re126FWCuII}2 also triggers intermolecular W122' → //*Re ET, and the Re(H126)(CO)3(dmp•-)//(W122•+)' charge-separated state decays to the ground state by ∼50 ns ReI(H126)(CO)3(dmp•-)+ → //(W122•+)' intermolecular charge recombination. Our findings shed light on the factors that control interfacial hole/electron hopping in protein complexes and on the role of aromatic amino acids in accelerating long-range electron transport.

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Why Do Most Aromatics Fail to Support Hole Hopping in the Cytochrome c Peroxidase-Cytochrome c Complex?

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