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Efficiently carrying out the oxygen reduction reaction (ORR) is critical for many applications in biology and chemistry, such as bioenergetics and fuel cells, respectively. In biology, this reaction is carried out by large, transmembrane oxidases such as heme-copper oxidases (HCOs) and cytochrome bdoxidases. Common to these oxidases is the presence of a glutamate residue next to the active site, but its precise role in regulating the oxidase activity remains unclear. To gain insight into its role, we herein report that incorporation of glutamate next to a designed heme-copper center in two biosynthetic models of HCOs improves O2binding affinity, facilitates protonation of reaction intermediates, and eliminates release of reactive oxygen species. High-resolution crystal structures of the models revealed extended, water-mediated hydrogen-bonding networks involving the glutamate. Electron paramagnetic resonance of the cryoreduced oxy-ferrous centers at cryogenic temperature followed by thermal annealing allowed observation of the key hydroperoxo intermediate that can be attributed to the hydrogen-bonding network. By demonstrating these important roles of glutamate in oxygen reduction biochemistry, this work offers deeper insights into its role in native oxidases, which may guide the design of more efficient artificial ORR enzymes or catalysts for applications such as fuel cells.

In the mycobacterial electron-transport chain, respiratory complex III passes electrons from menaquinol to complex IV, which in turn reduces oxygen, the terminal acceptor. Electron transfer is coupled to transmembrane proton translocation, thus establishing the electrochemical proton gradient that drives ATP synthesis. We isolated, biochemically characterized, and determined the structure of the obligate III2IV2supercomplex from Mycobacterium smegmatis, a model for Mycobacterium tuberculosis. The supercomplex has quinol:O2oxidoreductase activity without exogenous cytochrome cand includes a superoxide dismutase subunit that may detoxify reactive oxygen species produced during respiration. We found menaquinone bound in both the Qoand Qisites of complex III. The complex III-intrinsic diheme cytochrome ccsubunit, which functionally replaces both cytochrome c1and soluble cytochrome cin canonical electron-transport chains, displays two conformations: one in which it provides a direct electronic link to complex IV and another in which it serves as an electrical switch interrupting the connection.

Acidianus ambivalensis a hyperthermoacidophilic archaeon which grows optimally at ∼80°C and pH 2.5. The terminal oxidase of its respiratory system is a membrane-bound quinol oxidase (cytochrome aa3) which belongs to the heme–copper oxidase superfamily. One difference between this quinol oxidase and a majority of the other members of this family is that it lacks the highly-conserved glutamate (Glu(I-286), E. coliubiquinol oxidase numbering) which has been shown to play a central role in controlling the proton transfer during reaction of reduced oxidases with oxygen. In this study we have investigated the dynamics of the reaction of the reduced A. ambivalensquinol oxidase with O2. With the purified enzyme, two kinetic phases were observed with rate constants of 1.8⋅104s−1(at 1 mM O2, pH 7.8) and 3.7×103s−1, respectively. The first phase is attributed to binding of O2to heme a3and oxidation of both hemes forming the ‘peroxy’ intermediate. The second phase was associated with proton uptake from solution and it is attributed to formation of the ‘oxo-ferryl’ state, the final state in the absence of quinol. In the presence of bound caldariella quinol (QH2), heme awas re-reduced by QH2with a rate of 670 s−1, followed by transfer of the fourth electron to the binuclear center with a rate of 50 s−1. Thus, the results indicate that the quinol donates electrons to heme a, followed by intramolecular transfer to the binuclear center. Moreover, the overall electron and proton-transfer kinetics in the A. ambivalensquinol oxidase are the same as those in the E. coliubiquinol oxidase, which indicates that in the A. ambivalensenzyme a different pathway is used for proton transfer to the binuclear center and/or other protonatable groups in an equivalent pathway are involved. Potential candidates in that pathway are two glutamates at positions (I-80) and (I-83) in the A. ambivalensenzyme (corresponding to Met(I-116) and Val(I-119), respectively, in E. colicytochrome bo3).

Cytochrome c oxidase is a membrane‐bound redox‐driven proton pump. The coupling of the exergonic electron‐transfer reactions from cytochrome cto oxygen to proton translocation across the membrane requires control of internal electron‐ and proton‐transfer reactions. In this work, we focus on the kinetics of electron and proton transfer during those reaction steps that are coupled to proton pumping in cytochrome coxidase. The results show that during the first pumping step (peroxy → oxoferryl transition), proton transfer regulates intramolecular electron transfer. The proton transfer takes place in two steps: (1) Internal proton transfer from a protonatable group, proposed to be Glu(I‐286), in the so‐called D‐pathway, to an oxygen intermediate at the binuclear center (τ ≅ 100 μs); (2) Rapid re‐protonation of Glu(I‐286) from the bulk solution (τ < 100 μs). Only after proton uptake from solution the last (fourth) electron is transferred “one step closer” towards the binuclear center (from CuAto heme a). During the second proton‐pumping step (oxoferryl → oxidized), this electron is transferred to the binuclear center, linked to the uptake of a proton through the D‐pathway. The electron‐transfer rate displays a kinetic‐isotope effect (kH/kD) of 6 ± 1 (in a pH range in which the pH dependence of the rate is small), which indicates that the electron transfer is rate‐limited by the proton transfer. The entry into the D‐pathway (around Asp(I‐132)) is composed of a cluster of negatively‐charged amino acid residues together with a number of histidines, forming a so‐called proton‐collecting antenna designed to allow rapid protonation of groups within the proton‐transfer pathway.

The time course of absorbance changes following flash photolysis of the fully-reduced carboxycytochrome oxidase fromBacillus PS3 in the presence of O2 has been followed at 445, 550, 605, and 830 nm, and the results have been compared with the corresponding changes in bovine cytochrome oxidase. The PS3 enzyme has a covalently bound cytochromec subunit and the fully-reduced species therefore accommodates five electrons instead of four as in the bovine enzyme. In the bovine enzyme, following CO dissociation, four phases were observed with time constants of about 10 Μs, 30 Μs, 100 Μs, and 1 ms at 445 nm. The initial, 10-Μs absorbance change at 445 nm is similar in the two enzymes. The subsequent phases involving hemea and CuA are not seen in the PS3 enzyme at 445 nm, because these redox centers are re-reduced by the covalently bound cytochromec, as indicated by absorbance changes at 550 nm. A reaction scheme consistent with the experimental observations is presented. In addition, internal electron-transfer reactions in the absence of O2 were studied following flash-induced CO dissociation from the mixed-valence enzyme. Comparisons of the CO recombination rates in the mixed-valence and fully-reduced oxidases indicate that more electrons were transferred from hemea3 toa in PS3 oxidase compared to the bovine enzyme.

Wild-type and several mutants of cytochrome coxidase from Rhodobacter sphaeroideswere characterized by EPR spectroscopy. A pH-induced g12 signal, seen previously in mammalian cytochrome oxidase and assigned to the presence of a bridging car☐yl ligand in the bimetallic cytochrome a3-CuBsite, is found also in the bacterial enzyme. Mutation of glutamate-286 to glutamine inactivates the enzyme but does not affect this signal, demonstrating that the car☐yl group of this residue is not the bridging ligand. Three mutants, M106Q, located one helix turn below a histidine ligand to cytochrome a, and T352A as well as F391Q, located close to the bimetallic center, are shown to affect dramatically the low-spin heme signal of cytochrome a. These mutants are essentially inactive, suggesting that these three mutations result in alterations to cytochrome athat render the oxidase non-functional.

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 coxidase, 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 coxidase 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 O2reduction.

De novogene origination from nonfunctional DNA sequences was long assumed to be implausible. However, recent studies have shown that large fractions of genomic noncoding DNA are transcribed and translated, potentially generating new genes. Experimental validation of this process so far has been limited to comparative genomics, in vitroselections, or partial randomizations. Here, we describe selection of novel peptides in vivousing fully random synthetic expression libraries. The peptides confer aminoglycoside resistance by inserting into the bacterial membrane and thereby partly reducing membrane potential and decreasing drug uptake. Our results show that beneficial peptides can be selected from random sequence pools in vivoand support the idea that expression of noncoding sequences could spark the origination of new genes.