Somdatta Ghosh

Using a functional enzyme model to understand the chemistry behind hydrogen sulfide induced hibernation

The toxic gas H2S is produced by enzymes in the body. At moderate concentrations, H2S elicits physiological effects similar to hibernation. Herein, we describe experiments that imply that the phenomenon probably results from reversible inhibition of the enzyme cytochrome c oxidase (CcO), which reduces oxygen during respiration. A functional model of the oxygen-reducing site in CcO was used to explore the effects of H2S during respiration. Spectroscopic analyses showed that the model binds two molecules of H2S. The electro-catalytic reduction of oxygen is reversibly inhibited by H2S concentrations similar to those that induce hibernation. This phenomenon derives from a weak, reversible binding of H2S to the FeII porphyrin, which mimics heme a3 in CcOs active site. No inhibition of CcO is detected at lower H2S concentrations. Nevertheless, at lower concentrations, H2S could have other biological effects on CcO. For example, H2S rapidly reduces FeIII and CuII in both the oxidized form of this functional model and in CcO itself. H2S also reduces CcOs biological reductant, cytochrome c, which normally derives its reducing equivalents from food metabolism. Consequently, it is speculated that H2S might also serve as a source of electrons during periods of hibernation when food supplies are low.

A functional nitric oxide reductase model

A functional heme/nonheme nitric oxide reductase (NOR) model is presented. The fully reduced diiron compound reacts with two equivalents of NO leading to the formation of one equivalent of N2O and the bis-ferric product. NO binds to both heme Fe and nonheme Fe complexes forming individual ferrous nitrosyl species. The mixed-valence species with an oxidized heme and a reduced nonheme FeB does not show NO reduction activity. These results are consistent with a so-called “trans” mechanism for the reduction of NO by bacterial NOR.

Spectroscopic and Computational Studies of Nitrite Reductase: Proton Induced Electron Transfer and Backbonding Contributions to Reactivity

A combination of spectroscopy and DFT calculations has been used to define the geometric and electronic structure of the nitrite bound type 2 (T2) copper site at high and low pH in nitrite reductase from Rhodobacter sphaeroides. At high pH there is no electron transfer from reduced type 1 (T1) to the nitrite bound T2 copper, while protonation triggers T1 --> T2 electron transfer and generation of NO. The DFT calculated reaction coordinate for the N-O bond cleavage in nitrite reduction by the reduced T2 copper suggests that the process is best described as proton transfer triggering electron transfer. Bidentate nitrite binding to copper is calculated to play a major role in activating the reductive cleavage of the nitrite bond through backbonding combined with stabilization of the (-)OH product by coordination to the Cu(2+).

Catalytic Reduction of O2 by Cytochrome c Using a Synthetic Model of Cytochrome c Oxidase

Cytochrome c oxidase (CcO) catalyzes the four-electron reduction of oxygen to water, the one-electron reductant Cytochrome c (Cytc) being the source of electrons. Recently we reported a functional model of CcO that electrochemically catalyzes the four-electron reduction of O(2) to H(2)O (Collman et al. Science 2007, 315, 1565). The current paper shows that the same functional CcO model catalyzes the four-electron reduction of O(2) using the actual biological reductant Cytc in a homogeneous solution. Both single and steady-state turnover kinetics studies indicate that O(2) binding is rate-determining and that O-O bond cleavage and electron transfer from reduced Cytc to the oxidized model complex are relatively fast.

Thermodynamic equilibrium between blue and green copper sites and the role of the protein in controlling function

A combination of spectroscopies and density functional theory calculations indicate that there are large temperature-dependent absorption spectral changes present in green nitrite reductases (NiRs) due to a thermodynamic equilibrium between a green and a blue type 1 (T1) copper site. The axial methionine (Met) ligand is unconstrained in the oxidized NiRs, which results in an enthalpically favored (ΔH ≈4.6 kcal/mol) Met-bound green copper site at low temperatures, and an entropically favored (TΔS ≈4.5 kcal/mol, at room temperature) Met-elongated blue copper site at elevated temperatures. In contrast to the NiRs, the classic blue copper sites in plastocyanin and azurin show no temperature-dependent behavior, indicating that a single species is present at all temperatures. For these blue copper proteins, the polypeptide matrix opposes the gain in entropy that would be associated with the loss of the weak axial Met ligand at physiological temperatures by constraining its coordination to copper. The potential energy surfaces of Met binding indicate that it stabilizes the oxidized state more than the reduced state. This provides a mechanism to tune down the reduction potential of blue copper sites by >200 mV.

Recent applications of a synthetic model of cytochrome c oxidase: beyond functional modeling.

This account reports recent developments of a functional model for the active site of cytochrome c oxidase (CcO). This CcO mimic not only performs the selective four-electron reduction of oxygen to water but also catalytically reduces oxygen using the biological one-electron reductant, cytochrome c. This functional model has been used to understand other biological reactions of CcO, for example, the interaction between the gaseous hormone, NO, and CcO. A mechanism for inactivating NO-CcO complexes is found to involve a reaction between oxygen and Cu(B). Moreover, NO is shown to be capable of protecting CcO from toxic inhibitors such as CN(-) and CO. Finally, this functional CcO model has been used to show how H(2)S could induce hibernation by reversibly inhibiting the oxygen binding step involved in respiration.

Spectroscopic and Electronic Structure Studies of Phenolate Cu(II) Complexes: Phenolate Ring Orientation and Activation Related to Cofactor Biogenesis

A combination of spectroscopies and DFT calculations have been used to define the electronic structures of two crystallographically defined Cu(II)-phenolate complexes. These complexes differ in the orientation of the phenolate ring which results in different bonding interactions of the phenolate donor orbitals with the Cu(II), which are reflected in the very different spectroscopic properties of the two complexes. These differences in electronic structures lead to significant differences in DFT calculated reactivities with oxygen. These calculations suggest that oxygen activation via a Cu(I) phenoxyl ligand-to-metal charge transfer complex is highly endergonic (>50 kcal/mol), hence an unlikely pathway. Rather, the two-electron oxidation of the phenolate forming a bridging Cu(II) peroxoquinone complex is more favorable (11.3 kcal/mol). The role of the oxidized metal in mediating this two-electron oxidation of the coordinated phenolate and its relevance to the biogenesis of the covalently bound topa quinone in amine oxidase are discussed.

O2 reduction by a functional heme/nonheme bis-iron NOR model complex

O2 reactivity of a functional NOR model is investigated by using electrochemistry and spectroscopy. The electrochemical measurements using interdigitated electrodes show very high selectivity for 4e O2 reduction with minimal production of partially reduced oxygen species (PROS) under both fast and slow electron flux. Intermediates trapped at cryogenic temperatures and characterized by using resonance Raman spectroscopy under single-turnover conditions indicate that an initial bridging peroxide intermediate undergoes homolytic OO bond cleavage generating a trans heme/nonheme bis-ferryl intermediate. This bis ferryl species can oxygenate 2 equivalents of a reactive substrate.

Inhibition of Electrocatalytic O2 Reduction of Functional CcO Models by Competitive, Non-Competitive, and Mixed Inhibitors

Electrocatalytic reduction of O(2) by functional cytochrome C Oxidase (CcO) models is studied in the presence of several known inhibitors like CO, N(3)(-), CN(-), and NO(2)(-). These models successfully reproduce the inhibitions observed in CcO at similar concentrations reported for these inhibitors. Importantly, the data show very different electrochemical responses depending on the nature of the inhibitor, that is, competitive, non-competitive and mixed. Chemical models have been provided for these observed differences in the electrochemical behavior. Using the benchmark electrochemical behaviors for known inhibitors, the inhibition by NO(2)(-) is investigated. Electrochemical data suggests that NO(2)(-) acts as a competitive inhibitor at high concentrations. Spectroscopic data suggests that NO released during oxidation of the reduced catalyst in presence of excess NO(2)(-) is the source of the competitive inhibition by NO(2)(-). Presence of the distal Cu(B) lowers the inhibitory effect of CN(-) and NO(2)(-). While for CN(-) it weakens its binding affinity to the reduced complex by approximately 4.5 times, for NO(2)(-), it allows regeneration of the active catalyst from a catalytically inactive, air stable ferrous nitrosyl complex via a proposed superoxide mediated pathway.