The chemistry of the CuB site in cytochrome c oxidase and the importance of its unique His–Tyr bond

Helsinki Bioenergetics Group, Programme of Structural Biology and Biophysics, Institute of Biotechnology, University of Helsinki, Helsinki, Finland.
Biochimica et Biophysica Acta (Impact Factor: 4.66). 05/2009; 1787(4):221-33. DOI: 10.1016/j.bbabio.2009.01.002
Source: PubMed


The CuB metal center is at the core of the active site of the heme-copper oxidases, comprising a copper atom ligating three histidine residues one of which is covalently bonded to a tyrosine residue. Using quantum chemical methodology, we have studied the CuB site in several redox and ligand states proposed to be intermediates of the catalytic cycle. The importance of the His-Tyr crosslink was investigated by comparing energetics, charge, and spin distributions between systems with and without the crosslink. The His-Tyr bond was shown to decrease the proton affinity and increase the electron affinity of both Tyr-244 and the copper. A previously unnoticed internal electronic equilibrium between the copper atom and the tyrosine was observed, which seems to be coupled to the unique structure of the system. In certain states the copper and Tyr-244 compete for the unpaired electron, the localization of which is determined by the oxygenous ligand of the copper. This electronic equilibrium was found to be sensitive to the presence of a positive charge 10 A away from the center, simulating the effect of Lys-319 in the K-pathway of proton transfer. The combined results provide an explanation for why the heme-copper oxidases need two pathways of proton uptake, and why the K-pathway is active only in the second half of the reaction cycle.

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Available from: Dage Sundholm, Apr 24, 2014
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    • "Electrons donated by the reducing substrates are transferred intra-molecularly from heme a to heme a 3 and Cu B . In the fully reduced state, at 1.9 Å resolution, CcOX displays a trigonal planar coordination of Cu B by three histidine residues, one of which is covalently linked to a tyrosine residue of subunit I (Y244) thereby taking an important part in the O 2 reduction cycle [67] [68]. CcOX is targeted and inhibited by a number of small molecules/ions, such as CN "
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    ABSTRACT: BACKGROUND: The reactions between Complex IV (cytochrome c oxidase, CcOX) and nitric oxide (NO) were described in the early 60's. The perception, however, that NO could be responsible for physiological or pathological effects, including those on mitochondria, lags behind the 80's, when the identity of the endothelial derived relaxing factor (EDRF) and NO synthesis by the NO synthases were discovered. NO controls mitochondrial respiration, and cytotoxic as well as cytoprotective effects have been described. The depression of OXPHOS ATP synthesis has been observed, attributed to the inhibition of mitochondrial Complex I and IV particularly, found responsible of major effects. SCOPE OF REVIEW: The review is focused on CcOX and NO with some hints about pathophysiological implications. The reactions of interest are reviewed, with special attention to the molecular mechanisms underlying the effects of NO observed on cytochrome c oxidase, particularly during turnover with oxygen and reductants. MAJOR CONCLUSIONS AND GENERAL SIGNIFICANCE: The NO inhibition of CcOX is rapid and reversible and may occur in competition with oxygen. Inhibition takes place following two pathways leading to formation of either a relatively stable nitrosyl-derivative (CcOX-NO) of the enzyme reduced, or a more labile nitrite-derivative (CcOX-NO(2)(-)) of the enzyme oxidized, and during turnover. The pathway that prevails depends on the turnover conditions and concentration of NO and physiological substrates, cytochrome c and O(2). All evidence suggests that these parameters are crucial in determining the CcOX vs NO reaction pathway prevailing in vivo, with interesting physiological and pathological consequences for cells.
    Biochimica et Biophysica Acta 09/2011; 1817(4):610-9. DOI:10.1016/j.bbabio.2011.09.002 · 4.66 Impact Factor
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    • "The active site of CcO comprises two metal ions—a high-spin heme, heme a 3 , and a copper ion, Cu B (see Fig. 1B). Cu B is ligated to three histidines (His-240, His-290, His-291), one of which (His-240) is covalently linked by its side chain to a tyrosine residue (Tyr-244), which modifies the proton and electron affinities of the tyrosine [7] [8] [9] [10]. The reaction cycle of CcO begins by binding molecular oxygen to the fully reduced binuclear center (BNC) in state R. The formed species, known as state A [11] (see Fig. 1C), has an openshell singlet configuration and is best described as a ferric superoxide species [12] [13]. "
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    ABSTRACT: Cytochrome c oxidase (CcO) is the terminal enzyme of the respiratory chain. By reducing oxygen to water, it generates a proton gradient across the mitochondrial or bacterial membrane. Recently, two independent X-ray crystallographic studies ((Aoyama et al. Proc. Natl. Acad. Sci. USA 106 (2009) 2165-2169) and (Koepke et al. Biochim. Biophys. Acta 1787 (2009) 635-645)), suggested that a peroxide dianion might be bound to the active site of oxidized CcO. We have investigated this hypothesis by combining quantum chemical calculations with a re-refinement of the X-ray crystallographic data and optical spectroscopic measurements. Our data suggest that dianionic peroxide, superoxide, and dioxygen all form a similar superoxide species when inserted into a fully oxidized ferric/cupric binuclear site (BNC). We argue that stable peroxides are unlikely to be confined within the oxidized BNC since that would be expected to lead to bond splitting and formation of the catalytic P intermediate. Somewhat surprisingly, we find that binding of dioxygen to the oxidized binuclear site is weakly exergonic, and hence, the observed structure might have resulted from dioxygen itself or from superoxide generated from O(2) by the X-ray beam. We show that the presence of O(2) is consistent with the X-ray data. We also discuss how other structures, such as a mixture of the aqueous species (H(2)O+OH(-) and H(2)O) and chloride fit the experimental data.
    Biochimica et Biophysica Acta 07/2011; 1807(7):769-78. DOI:10.1016/j.bbabio.2010.12.016 · 4.66 Impact Factor
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    • "In this context, quantum chemical vibrational analysis of model complexes can help the interpretation of experimental results [34] [35]. The role of the tyrosine cross-link has also been addressed by quantum chemical calculations [36] [37], and finally a few quantum chemical calculations have been made trying to investigate one of the proposed proton pathways [38] "
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    ABSTRACT: Recent developments of quantum chemical methods have made it possible to tackle crucial questions in bioenergetics. The most important systems, cytochrome c oxidase in cellular respiration and photosystem II (PSII) in photosynthesis will here be used as examples to illustrate the power of the quantum chemical tools. One main contribution from quantum chemistry is to put mechanistic suggestions onto an energy scale. Accordingly, free energy profiles can be constructed both for reduction of molecular oxygen in cytochrome c oxidase and water oxidation in PSII, including O-O bond cleavage and formation, and also proton pumping in cytochrome c oxidase. For the construction of the energy diagrams, the computational results sometimes have to be combined with experimental information, such as reduction potentials and rate constants for individual steps in the reactions.
    Biochimica et Biophysica Acta 10/2009; 1797(2):129-42. DOI:10.1016/j.bbabio.2009.10.004 · 4.66 Impact Factor
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