Pia Ädelroth

Bio: Pia Ädelroth is an academic researcher from Stockholm University. The author has contributed to research in topics: Cytochrome c oxidase & Electron transfer. The author has an hindex of 32, co-authored 98 publications receiving 3056 citations. Previous affiliations of Pia Ädelroth include University of California, San Diego & University of East Anglia.

A mechanistic principle for proton pumping by cytochrome c oxidase

Abstract: In aerobic organisms, cellular respiration involves electron transfer to oxygen through a series of membrane-bound protein complexes. The process maintains a transmembrane electrochemical proton gradient that is used, for example, in the synthesis of ATP. In mitochondria and many bacteria, the last enzyme complex in the electron transfer chain is cytochrome c oxidase (CytcO), which catalyses the four-electron reduction of O2 to H2O using electrons delivered by a water-soluble donor, cytochrome c. The electron transfer through CytcO, accompanied by proton uptake to form H2O drives the physical movement (pumping) of four protons across the membrane per reduced O2. So far, the molecular mechanism of such proton pumping driven by electron transfer has not been determined in any biological system. Here we show that proton pumping in CytcO is mechanistically coupled to proton transfer to O2 at the catalytic site, rather than to internal electron transfer. This scenario suggests a principle by which redox-driven proton pumps might operate and puts considerable constraints on possible molecular mechanisms by which CytcO translocates protons.

Pathways of Proton Transfer in Cytochrome c Oxidase

Abstract: During the last few years our knowledge of the structure and function of heme copper oxidases has greatly profited from the use of site-directed mutagenesis in combination with biophysical techniques. This, together with the recently-determined crystal structures of cytochrome c oxidase, has now made it possible to design experiments aimed at targeting specific pump mechanisms. Here, we summarize results from our recent kinetic studies of electron and proton-transfer reactions in wild-type and mutant forms of cytochrome c oxidase from Rhodobacter sphaeroides. These studies have made it possible to identify amino acid residues involved in proton transfer during specific reaction steps and provide a basis for discussion of mechanisms of electron and proton transfer in terminal oxidases. The results indicate that the pathway through K(I-362)/T(I-359), but not through D(I-132)/E(I-286), is used for proton transfer to a protonatable group interacting electrostatically with heme a3, i.e., upon reduction of the binuclear center. The pathway through D(I-132)/E(I-286) is used for uptake of pumped and substrate protons during the pumping steps during O2 reduction.

Glutamate 286 in cytochrome aa3 from Rhodobacter sphaeroides is involved in proton uptake during the reaction of the fully-reduced enzyme with dioxygen

Abstract: The reaction with dioxygen of solubilized fully-reduced wild-type and EQ(I-286) (exchange of glutamate 286 of subunit I for glutamine) mutant cytochrome c oxidase from Rhodobacter sphaeroides has been studied using the flow-flash technique in combination with optical absorption spectroscopy. Proton uptake was measured using a pH-indicator dye. In addition, internal electron-transfer reactions were studied in the absence of oxygen. Glutamate 286 is found in a proton pathway proposed to be used for pumped protons from the crystal structure of cytochrome c oxidase from Paracoccus denitrificans [Iwata et al. (1995) Nature 376, 660-669; E278 in P.d. numbering]. It is the residue closest to the oxygen-binding binuclear center that is clearly a part of the pathway. The results show that the wild-type enzyme becomes fully oxidized in a few milliseconds at pH 7.4 and displays a biphasic proton uptake from the medium. In the EQ(I-286) mutant enzyme, electron transfer after formation of the peroxy intermediate is impaired, CuA remains reduced, and no protons are taken up from the medium. Thus, the results suggest that E(I-286) is necessary for proton uptake after formation of the peroxy intermediate and transfer of the fourth electron to the binuclear center. The results also indicate that the proton uptake associated with formation of the ferryl intermediate controls the electron transfer from CuA to heme a.

On the role of the K-proton transfer pathway in cytochrome c oxidase

Abstract: Cytochrome c oxidase is a membrane-bound enzyme that catalyzes the four-electron reduction of oxygen to water. This highly exergonic reaction drives proton pumping across the membrane. One of the key questions associated with the function of cytochrome c oxidase is how the transfer of electrons and protons is coupled and how proton transfer is controlled by the enzyme. In this study we focus on the function of one of the proton transfer pathways of the R. sphaeroides enzyme, the so-called K-proton transfer pathway (containing a highly conserved Lys(I-362) residue), leading from the protein surface to the catalytic site. We have investigated the kinetics of the reaction of the reduced enzyme with oxygen in mutants of the enzyme in which a residue [Ser(I-299)] near the entry point of the pathway was modified with the use of site-directed mutagenesis. The results show that during the initial steps of oxygen reduction, electron transfer to the catalytic site (to form the “peroxy” state, Pr) requires charge compensation through the proton pathway, but no proton uptake from the bulk solution. The charge compensation is proposed to involve a movement of the K(I-362) side chain toward the binuclear center. Thus, in contrast to what has been assumed previously, the results indicate that the K-pathway is used during oxygen reduction and that K(I-362) is charged at pH ≈ 7.5. The movement of the Lys is proposed to regulate proton transfer by “shutting off” the protonic connectivity through the K-pathway after initiation of the O2 reduction chemistry. This “shutoff” prevents a short-circuit of the proton-pumping machinery of the enzyme during the subsequent reaction steps.

Role of the pathway through K(I-362) in proton transfer in cytochrome c oxidase from R. sphaeroides.

Abstract: In this study we have combined the use of site-directed mutants with time-resolved optical absorption spectroscopy to investigate the role of the protonatable subunit-I residues lysine-362 (K(I-362)) and threonine-359 (T(I-359)) in cytochrome c oxidase from Rhodobacter sphaeroides in electron and proton transfer. These residues have been proposed to be part of a proton-transfer pathway in cytochrome oxidases from Paracoccus denitrificans and bovine heart. Mutation of K(I-362) and T(I-359) to methionine and alanine, respectively, results in reduction of the overall turnover activities to <2% and ∼35%, respectively, of those in the wild-type enzyme. The results show that in the absence of dioxygen, electron transfer between hemes a3 and a with a time constant of ∼3 μs, not coupled to protonation reactions, is not affected in the mutant enzymes. However, the slower electron transfer between hemes a3 and a, coupled to proton release with a time constant of ∼3 ms (at pH 9.0) is impaired in the KM(I-362) and TA...

Proton-Coupled Electron Transfer

Abstract: ▪ Abstract Proton-coupled electron transfer (PCET) is an important mechanism for charge transfer in a wide variety of systems including biology- and materials-oriented venues. We review several are...

Water-splitting chemistry of photosystem II.

Abstract: Life on earth is almost entirely solar-powered. We can get some idea of the enormous quantity of energy received from the sun by noting that during daylight hours, the sun provides several thousand times more power to the surface of the U.S.A. than is produced by all of the nation’s electrical power stations. 1,2 Around 50% of the radiation that reaches the earth’s surface, roughly the visible region, is of a frequency useful to photosynthetic organisms. Oxygenic photosynthetic organisms convert this radiation into chemical energy, in the form of carbohydrate and dioxygen, at an optimal efficiency of something like 25%. 3 These products together sustain the rest of aerobic life, with carbohydrate acting as a source of high-energy electrons and dioxygen providing a lower-energy destination for these electrons. The overall equation of oxygenic photosynthesis is given in eq 1, where (CH2O) represents carbohydrate:

Copper Active Sites in Biology

Abstract: Based on its generally accessible I/II redox couple and bioavailability, copper plays a wide variety of roles in nature that mostly involve electron transfer (ET), O2 binding, activation and reduction, NO2− and N2O reduction and substrate activation. Copper sites that perform ET are the mononuclear blue Cu site that has a highly covalent CuII-S(Cys) bond and the binuclear CuA site that has a Cu2S(Cys)2 core with a Cu-Cu bond that keeps the site delocalized (Cu(1.5)2) in its oxidized state. In contrast to inorganic Cu complexes, these metalloprotein sites transfer electrons rapidly often over long distances, as has been previously reviewed.1–4 Blue Cu and CuA sites will only be considered here in their relation to intramolecular ET in multi-center enzymes. The focus of this review is on the Cu enzymes (Figure 1). Many are involved in O2 activation and reduction, which has mostly been thought to involve at least two electrons to overcome spin forbiddenness and the low potential of the one electron reduction to superoxide (Figure 2).5,6 Since the Cu(III) redox state has not been observed in biology, this requires either more than one Cu center or one copper and an additional redox active organic cofactor. The latter is formed in a biogenesis reaction of a residue (Tyr) that is also Cu catalyzed in the first turnover of the protein. Recently, however, there have been a number of enzymes suggested to utilize one Cu to activate O2 by 1e− reduction to form a Cu(II)-O2•− intermediate (an innersphere redox process) and it is important to understand the active site requirements to drive this reaction. The oxidases that catalyze the 4e−reduction of O2 to H2O are unique in that they effectively perform this reaction in one step indicating that the free energy barrier for the second two-electron reduction of the peroxide product of the first two-electron step is very low. In nature this requires either a trinuclear Cu cluster (in the multicopper oxidases) or a Cu/Tyr/Heme Fe cluster (in the cytochrome oxidases). The former accomplishes this with almost no overpotential maximizing its ability to oxidize substrates and its utility in biofuel cells, while the latter class of enzymes uses the excess energy to pump protons for ATP synthesis. In bacterial denitrification, a mononuclear Cu center catalyzes the 1e- reduction of nitrite to NO while a unique µ4S2−Cu4 cluster catalyzes the reduction of N2O to N2 and H2O, a 2e− process yet requiring 4Cu’s. Finally there are now several classes of enzymes that utilize an oxidized Cu(II) center to activate a covalently bound substrate to react with O2. Figure 1 Copper active sites in biology. Figure 2 Latimer Diagram for Oxygen Reduction at pH = 7.0 Adapted from References 5 and 6. This review presents in depth discussions of all these classes of Cu enzymes and the correlations within and among these classes. For each class we review our present understanding of the enzymology, kinetics, geometric structures, electronic structures and the reaction mechanisms these have elucidated. While the emphasis here is on the enzymology, model studies have significantly contributed to our understanding of O2 activation by a number of Cu enzymes and are included in appropriate subsections of this review. In general we will consider how the covalency of a Cu(II)–substrate bond can activate the substrate for its spin forbidden reaction with O2, how in binuclear Cu enzymes the exchange coupling between Cu’s overcomes the spin forbiddenness of O2 binding and controls electron transfer to O2 to direct catalysis either to perform two e− electrophilic aromatic substitution or 1e− H-atom abstraction, the type of oxygen intermediate that is required for H-atom abstraction from the strong C-H bond of methane (104 kcal/mol) and how the trinuclear Cu cluster and the Cu/Tyr/Heme Fe cluster achieve their very low barriers for the reductive cleavage of the O-O bond. Much of the insight available into these mechanisms in Cu biochemistry has come from the application of a wide range of spectroscopies and the correlation of spectroscopic results to electronic structure calculations. Thus we start with a tutorial on the different spectroscopic methods utilized to study mononuclear and multinuclear Cu enzymes and their correlations to different levels of electronic structure calculations.

Structure and function of photosystems i and ii

Abstract: Oxygenic photosynthesis, the principal converter of sunlight into chemical energy on earth, is catalyzed by four multi-subunit membrane-protein complexes: photosystem I (PSI), photosystem II (PSII), the cytochrome b6f complex, and F-ATPase PSI generates the most negative redox potential in nature and largely determines the global amount of enthalpy in living systems PSII generates an oxidant whose redox potential is high enough to enable it to oxidize H2O, a substrate so abundant that it assures a practically unlimited electron source for life on earth During the last century, the sophisticated techniques of spectroscopy, molecular genetics, and biochemistry were used to reveal the structure and function of the two photosystems The new structures of PSI and PSII from cyanobacteria, algae, and plants has shed light not only on the architecture and mechanism of action of these intricate membrane complexes, but also on the evolutionary forces that shaped oxygenic photosynthesis