Enzymatic cofactor regeneration systems: A new perspective on efficiency assessment.

Nowadays, the specificity of enzymatic processes makes them more and more important every year, and their usage on an industrial scale seems to be necessary. Enzymatic cofactors, however, play a crucial part in the prospective applications of enzymes, because they are indispensable for conducting highly effective biocatalytic activities. Due to the relatively high cost of these compounds and their consumption during the processes carried out, it has become crucial to develop systems for cofactor regeneration. Therefore, in this review, an attempt was made to summarize current knowledge on enzymatic regeneration methods, which are characterized by high specificity, non-toxicity and reported to be highly efficient. The regeneration of cofactors, such as nicotinamide dinucleotides, coenzyme A, adenosine 5'-triphosphate and flavin nucleotides, which are necessary for the proper functioning of a large number of enzymes, is discussed, as well as potential directions for further development of these systems are highlighted. This review discusses a range of highly effective cofactor regeneration systems along with the productive synthesis of many useful chemicals, including the simultaneous renewal of several cofactors at the same time. Additionally, the impact of the enzyme immobilization process on improving the stability and the potential for multiple uses of the developed cofactor regeneration systems was also presented. Moreover, an attempt was made to emphasize the importance of the presented research, as well as the identification of research gaps, which mainly result from the lack of available literature on this topic.

Function of Molybdenum Insertases.

For most organisms molybdenum is essential for life as it is found in the active site of various vitally important molybdenum dependent enzymes (Mo-enzymes). Here, molybdenum is bound to a pterin derivative called molybdopterin (MPT), thus forming the molybdenum cofactor (Moco). Synthesis of Moco involves the consecutive action of numerous enzymatic reaction steps, whereby molybdenum insertases (Mo-insertases) catalyze the final maturation step, i.e., the metal insertion reaction yielding Moco. This final maturation step is subdivided into two partial reactions, each catalyzed by a distinctive Mo-insertase domain. Initially, MPT is adenylylated by the Mo-insertase G-domain, yielding MPT-AMP which is used as substrate by the E-domain. This domain catalyzes the insertion of molybdate into the MPT dithiolene moiety, leading to the formation of Moco-AMP. Finally, the Moco-AMP phosphoanhydride bond is cleaved by the E-domain to liberate Moco from its synthesizing enzyme. Thus formed, Moco is physiologically active and may be incorporated into the different Mo-enzymes or bind to carrier proteins instead.

Unveiling the mechanisms and biosynthesis of a novel nickel-pincer enzyme.

The nickel-pincer nucleotide (NPN) coenzyme, a substituted pyridinium mononucleotide that tri-coordinates nickel, was first identified covalently attached to a lysine residue in the LarA protein of lactate racemase. Starting from nicotinic acid adenine dinucleotide, LarB carboxylates C5 of the pyridinium ring and hydrolyzes the phosphoanhydride, LarE converts the C3 and C5 carboxylates to thiocarboxylates, and LarC incorporates nickel to form a C-Ni and two S-Ni bonds, during the biosynthesis of this cofactor. LarB uses a novel carboxylation mechanism involving the transient formation of a cysteinyl-pyridinium adduct. Depending on the source of the enzyme, LarEs either catalyze a sacrificial sulfur transfer from a cysteinyl side chain resulting in the formation of dehydroalanine or they utilize a [4Fe-4S] cluster bound by three cysteine residues to accept and transfer a non-core sulfide atom. LarC is a CTP-dependent enzyme that cytidinylylates its substrate, adds nickel, then hydrolyzes the product to release NPN and CMP. Homologs of the four lar genes are widely distributed in microorganisms, with some species containing multiple copies of larA whereas others lack this gene, consistent with the cofactor serving other functions. Several LarA-like proteins were shown to catalyze racemase or epimerase activities using 2-hydroxyacid substrates other than lactic acid. Thus, lactate racemase is the founding member of a large family of NPN-containing enzymes.

A complete biomimetic iron-sulfur cubane redox series.

Synthetic iron-sulfur cubanes are models for biological cofactors, which are essential to delineate oxidation states in the more complex enzymatic systems. However, a complete series of [Fe4S4]n complexes spanning all redox states accessible by 1-electron transformations of the individual iron atoms (n = 0-4+) has never been prepared, deterring the methodical comparison of structure and spectroscopic signature. Here, we demonstrate that the use of a bulky arylthiolate ligand promoting the encapsulation of alkali-metal cations in the vicinity of the cubane enables the synthesis of such a series. Characterization by EPR, 57Fe Mössbauer spectroscopy, UV-visible electronic absorption, variable-temperature X-ray diffraction analysis, and cyclic voltammetry reveals key trends for the geometry of the Fe4S4 core as well as for the Mössbauer isomer shift, which both correlate systematically with oxidation state. Furthermore, we confirm the S = 4 electronic ground state of the most reduced member of the series, [Fe4S4]0, and provide electrochemical evidence that it is accessible within 0.82 V from the [Fe4S4]2+ state, highlighting its relevance as a mimic of the nitrogenase iron protein cluster.

The periodic table of ribonucleotide reductases.

In most organisms, transition metal ions are necessary cofactors of ribonucleotide reductase (RNR), the enzyme responsible for biosynthesis of the 2'-deoxynucleotide building blocks of DNA. The metal ion generates an oxidant for an active site cysteine (Cys), yielding a thiyl radical that is necessary for initiation of catalysis in all RNRs. Class I enzymes, widespread in eukaryotes and aerobic microbes, share a common requirement for dioxygen in assembly of the active Cys oxidant and a unique quaternary structure, in which the metallo- or radical-cofactor is found in a separate subunit, ß, from the catalytic α subunit. The first class I RNRs, the class Ia enzymes, discovered and characterized more than 30 years ago, were found to use a diiron(III)-tyrosyl-radical Cys oxidant. Although class Ia RNRs have historically served as the model for understanding enzyme mechanism and function, more recently, remarkably diverse bioinorganic and radical cofactors have been discovered in class I RNRs from pathogenic microbes. These enzymes use alternative transition metal ions, such as manganese, or posttranslationally installed tyrosyl radicals for initiation of ribonucleotide reduction. Here we summarize the recent progress in discovery and characterization of novel class I RNR radical-initiating cofactors, their mechanisms of assembly, and how they might function in the context of the active class I holoenzyme complex.

Therapeutic nanoreactors for detoxification of xenobiotics: Concepts, challenges and biotechnological trends with special emphasis to organophosphate bioscavenging.

The introduction of enzyme nanoreactors in medicine is relatively new. However, this technology has already been experimentally successful in cancer treatments, struggle against toxicity of reactive oxygen species in inflammatory processes, detoxification of drugs and xenobiotics, and correction of metabolic and genetic defects by using encapsulated enzymes, acting in single or cascade reactions. Biomolecules, e.g. enzymes, antibodies, reactive proteins capable of inactivating toxicants in the body are called bioscavengers. In this review, we focus on enzyme-containing nanoreactors for in vivo detoxification of organophosphorous compounds (OP) to be used for prophylaxis and post-exposure treatment of OP poisoning. A particular attention is devoted to bioscavenger-containing injectable nanoreactors operating in the bloodstream. The nanoreactor concept implements single or multiple enzymes and cofactors co-encapsulated in polymeric semi-permeable nanocontainers. Thus, the detoxification processes take place in a confined space containing highly concentrated bioscavengers. The article deals with historical and theoretical backgrounds about enzymatic detoxification of OPs in nanoreactors, nanoreactor polymeric enveloppes, realizations and advantages over other approaches using bioscavengers.

Mechanism of molybdate insertion into pterin-based molybdenum cofactors.

The molybdenum cofactor (Moco) is found in the active site of numerous important enzymes that are critical to biological processes. The bidentate ligand that chelates molybdenum in Moco is the pyranopterin dithiolene (molybdopterin, MPT). However, neither the mechanism of molybdate insertion into MPT nor the structure of Moco prior to its insertion into pyranopterin molybdenum enzymes is known. Here, we report this final maturation step, where adenylated MPT (MPT-AMP) and molybdate are the substrates. X-ray crystallography of the Arabidopsis thaliana Mo-insertase variant Cnx1E S269D D274S identified adenylated Moco (Moco-AMP) as an unexpected intermediate in this reaction sequence. X-ray absorption spectroscopy revealed the first coordination sphere geometry of Moco trapped in the Cnx1E active site. We have used this structural information to deduce a mechanism for molybdate insertion into MPT-AMP. Given their high degree of structural and sequence similarity, we suggest that this mechanism is employed by all eukaryotic Mo-insertases.

Characterization of an aminotransferase from Acanthamoeba polyphaga Mimivirus.

Acanthamoeba polyphaga Mimivirus, a complex virus that infects amoeba, was first reported in 2003. It is now known that its DNA genome encodes for nearly 1,000 proteins including enzymes that are required for the biosynthesis of the unusual sugar 4-amino-4,6-dideoxy-d-glucose, also known as d-viosamine. As observed in some bacteria, the pathway for the production of this sugar initiates with a nucleotide-linked sugar, which in the Mimivirus is thought to be UDP-d-glucose. The enzyme required for the installment of the amino group at the C-4' position of the pyranosyl moiety is encoded in the Mimivirus by the L136 gene. Here, we describe a structural and functional analysis of this pyridoxal 5'-phosphate-dependent enzyme, referred to as L136. For this analysis, three high-resolution X-ray structures were determined: the wildtype enzyme/pyridoxamine 5'-phosphate/dTDP complex and the site-directed mutant variant K185A in the presence of either UDP-4-amino-4,6-dideoxy-d-glucose or dTDP-4-amino-4,6-dideoxy-d-glucose. Additionally, the kinetic parameters of the enzyme utilizing either UDP-d-glucose or dTDP-d-glucose were measured and demonstrated that L136 is efficient with both substrates. This is in sharp contrast to the structurally related DesI from Streptomyces venezuelae, whose three-dimensional architecture was previously reported by this laboratory. As determined in this investigation, DesI shows a profound preference in its catalytic efficiency for the dTDP-linked sugar substrate. This difference can be explained in part by a hydrophobic patch in DesI that is missing in L136. Notably, the structure of L136 reported here represents the first three-dimensional model for a virally encoded PLP-dependent enzyme and thus provides new information on sugar aminotransferases in general.

Functional and structural characterization of a flavoprotein monooxygenase essential for biogenesis of tryptophylquinone cofactor.

Bioconversion of peptidyl amino acids into enzyme cofactors is an important post-translational modification. Here, we report a flavoprotein, essential for biosynthesis of a protein-derived quinone cofactor, cysteine tryptophylquinone, contained in a widely distributed bacterial enzyme, quinohemoprotein amine dehydrogenase. The purified flavoprotein catalyzes the single-turnover dihydroxylation of the tryptophylquinone-precursor, tryptophan, in the protein substrate containing triple intra-peptidyl crosslinks that are pre-formed by a radical S-adenosylmethionine enzyme within the ternary complex of these proteins. Crystal structure of the peptidyl tryptophan dihydroxylase reveals a large pocket that may dock the protein substrate with the bound flavin adenine dinucleotide situated close to the precursor tryptophan. Based on the enzyme-protein substrate docking model, we propose a chemical reaction mechanism of peptidyl tryptophan dihydroxylation catalyzed by the flavoprotein monooxygenase. The diversity of the tryptophylquinone-generating systems suggests convergent evolution of the peptidyl tryptophan-derived cofactors in different proteins.

Seesaw conformations of Npl4 in the human p97 complex and the inhibitory mechanism of a disulfiram derivative.

p97, also known as valosin-containing protein (VCP) or Cdc48, plays a central role in cellular protein homeostasis. Human p97 mutations are associated with several neurodegenerative diseases. Targeting p97 and its cofactors is a strategy for cancer drug development. Despite significant structural insights into the fungal homolog Cdc48, little is known about how human p97 interacts with its cofactors. Recently, the anti-alcohol abuse drug disulfiram was found to target cancer through Npl4, a cofactor of p97, but the molecular mechanism remains elusive. Here, using single-particle cryo-electron microscopy (cryo-EM), we uncovered three Npl4 conformational states in complex with human p97 before ATP hydrolysis. The motion of Npl4 results from its zinc finger motifs interacting with the N domain of p97, which is essential for the unfolding activity of p97. In vitro and cell-based assays showed that the disulfiram derivative bis-(diethyldithiocarbamate)-copper (CuET) can bypass the copper transporter system and inhibit the function of p97 in the cytoplasm by releasing cupric ions under oxidative conditions, which disrupt the zinc finger motifs of Npl4, locking the essential conformational switch of the complex.

In silico screening and molecular dynamics simulation of deleterious PAH mutations responsible for phenylketonuria genetic disorder.

Phenylketonuria (PKU) is a genetic disorder that if left untreated can lead to behavioral problems, epilepsy, and even mental retardation. PKU results from mutations within the phenylalanine-4-hydroxylase (PAH) gene that encodes for the PAH protein. The study of all PAH causing mutations is improbable using experimental techniques. In this study, a collection of in silico resources, sorting intolerant from tolerant, Polyphen-2, PhD-SNP, and MutPred were used to identify possible pathogenetic and deleterious PAH non-synonymous single nucleotide polymorphisms (nsSNPs). We identified two variants of PAH, I65N and L311P, to be the most deleterious and disease causing nsSNPs. Molecular dynamics (MD) simulations were carried out to characterize these point mutations on the atomic level. MD simulations revealed increased flexibility and a decrease in the hydrogen bond network for both mutants compared to the native protein. Free energy calculations using the MM/GBSA approach found that BH4 , a drug-based therapy for PKU patients, had a higher binding affinity for I65N and L311P mutants compared to the wildtype protein. We also identify important residues in the BH4 binding pocket that may be of interest for the rational drug design of other PAH drug-based therapies. Lastly, free energy calculations also determined that the I65N mutation may impair the dimerization of the N-terminal regulatory domain of PAH.

Ca2+ as cofactor of the mitochondrial H+ -translocating F1 FO -ATP(hydrol)ase.

The mitochondrial F1 FO -ATPase in the presence of the natural cofactor Mg2+ acts as the enzyme of life by synthesizing ATP, but it can also hydrolyze ATP to pump H+ . Interestingly, Mg2+ can be replaced by Ca2+ , but only to sustain ATP hydrolysis and not ATP synthesis. When Ca2+ inserts in F1 , the torque generation built by the chemomechanical coupling between F1 and the rotating central stalk was reported as unable to drive the transmembrane H+ flux within FO . However, the failed H+ translocation is not consistent with the oligomycin-sensitivity of the Ca2+ -dependent F1 FO -ATP(hydrol)ase. New enzyme roles in mitochondrial energy transduction are suggested by recent advances. Accordingly, the structural F1 FO -ATPase distortion driven by ATP hydrolysis sustained by Ca2+ is consistent with the permeability transition pore signal propagation pathway. The Ca2+ -activated F1 FO -ATPase, by forming the pore, may contribute to dissipate the transmembrane H+ gradient created by the same enzyme complex.

Round, round we go - strategies for enzymatic cofactor regeneration.

Covering: up to the beginning of 2020Enzymes depending on cofactors are essential in many biosynthetic pathways of natural products. They are often involved in key steps: catalytic conversions that are difficult to achieve purely with synthetic organic chemistry. Hence, cofactor-dependent enzymes have great potential for biocatalysis, on the condition that a corresponding cofactor regeneration system is available. For some cofactors, these regeneration systems require multiple steps; such complex enzyme cascades/multi-enzyme systems are (still) challenging for in vitro biocatalysis. Further, artificial cofactor analogues have been synthesised that are more stable, show an altered reaction range, or act as inhibitors. The development of bio-orthogonal systems that can be used for the production of modified natural products in vivo is an ongoing challenge. In light of the recent progress in this field, this review aims to provide an overview of general strategies involving enzyme cofactors, cofactor analogues, and regeneration systems; highlighting the current possibilities for application of enzymes using some of the most common cofactors.

Model Substrate/Inactivation Reactions for MoaA and Ribonucleotide Reductases: Loss of Bromo, Chloro, or Tosylate Groups from C2 of 1,5-Dideoxyhomoribofuranoses upon Generation of an α-Oxy Radical at C3.

We report studies on radical-initiated fragmentations of model 1,5-dideoxyhomoribofuranose derivatives with bromo, chloro, and tosyloxy substituents on C2. The effects of stereochemical inversion at C2 were probed with the corresponding arabino epimers. In all cases, the elimination of bromide, chloride, and tosylate anions occurred when the 3-hydroxyl group was unprotected. The isolation of deuterium-labeled furanone products established heterolytic cleavage followed by the transfer of deuterium from labeled tributylstannane. In contrast, 3-O-methyl derivatives underwent the elimination of bromine or chlorine radicals to give the 2,3-alkene with no incorporation of label in the methyl vinyl ether. More drastic fragmentation occurred with both of the 3-O-methyl-2-tosyloxy epimers to give an aromatized furan derivative with no deuterium label. Contrasting results observed with the present anhydroalditol models relative to our prior studies with analogously substituted nucleoside models have demonstrated that insights from biomimetic chemical reactions can provide illumination of mechanistic pathways employed by ribonucleotide reductases (RNRs) and the MoaA enzyme involved in the biosynthesis of molybdopterin.

Bond-valence analyses of the crystal structures of FeMo/V cofactors in FeMo/V proteins.

The bond-valence method has been used for valence calculations of FeMo/V cofactors in FeMo/V proteins using 51 crystallographic data sets of FeMo/V proteins from the Protein Data Bank. The calculations show molybdenum(III) to be present in MoFe7S9C(Cys)(HHis)[R-(H)homocit] (where H4homocit is homocitric acid, HCys is cysteine and HHis is histidine) in FeMo cofactors, while vanadium(III) with a more reduced iron complement is obtained for FeV cofactors. Using an error analysis of the calculated valences, it was found that in FeMo cofactors Fe1, Fe6 and Fe7 can be unambiguously assigned as iron(III), while Fe2, Fe3, Fe4 and Fe5 show different degrees of mixed valences for the individual Fe atoms. For the FeV cofactors in PDB entry 5n6y, Fe4, Fe5 and Fe6 correspond to iron(II), iron(II) and iron(III), respectively, while Fe1, Fe2, Fe3 and Fe7 exhibit strongly mixed valences. Special situations such as CO-bound and selenium-substituted FeMo cofactors and O(N)H-bridged FeV cofactors are also discussed and suggest rearrangement of the electron configuration on the substitution of the bridging S atoms.

Biologically generated carbon dioxide: nature's versatile chemical strategies for carboxy lyases.

Covering: up to 2019Metabolic production of CO2 is natural product chemistry on a mammoth scale. Just counting humans, among all other respiring organisms, the seven billion people on the planet exhale about 3 billion tons of CO2 per year. Essentially all of the biogenic CO2 arises by action of discrete families of decarboxylases. The mechanistic routes to CO2 release from carboxylic acid metabolites vary with the electronic demands and structures of specific substrates and illustrate the breadth of chemistry employed for C-COO (C-C bond) disconnections. Most commonly decarboxylated are α-keto acid and ß-keto acid substrates, the former requiring thiamin-PP as cofactor, the latter typically cofactor-free. The extensive decarboxylation of amino acids, e.g. to neurotransmitter amines, is synonymous with the coenzyme form of vitamin B6, pyridoxal-phosphate, although covalent N-terminal pyruvamide residues serve in some amino acid decarboxylases. All told, five B vitamins (B1, B2, B3, B6, B7), ATP, S-adenosylmethionine, manganese and zinc ions are pressed into service for specific decarboxylase catalyses. There are additional cofactor-independent decarboxylases that operate by distinct chemical routes. Finally, while most decarboxylases use heterolytic ionic mechanisms, a small number of decarboxylases carry out radical pathways.

Anion Binding and Oxidative Modification at the Molybdenum Cofactor of Formate Dehydrogenase from Rhodobacter capsulatus Studied by X-ray Absorption Spectroscopy.

Formate dehydrogenase (FDH) enzymes are versatile catalysts for CO2 conversion. The FDH from Rhodobacter capsulatus contains a molybdenum cofactor with the dithiolene functions of two pyranopterin guanine dinucleotide molecules, a conserved cysteine, and a sulfido group bound at Mo(VI). In this study, we focused on metal oxidation state and coordination changes in response to exposure to O2, inhibitory anions, and redox agents using X-ray absorption spectroscopy (XAS) at the Mo K-edge. Differences in the oxidative modification of the bis-molybdopterin guanine dinucleotide (bis-MGD) cofactor relative to samples prepared aerobically without inhibitor, such as variations in the relative numbers of sulfido (Mo═S) and oxo (Mo═O) bonds, were observed in the presence of azide (N3-) or cyanate (OCN-). Azide provided best protection against O2, resulting in a quantitatively sulfurated cofactor with a displaced cysteine ligand and optimized formate oxidation activity. Replacement of the cysteine ligand by a formate (HCO2-) ligand at the molybdenum in active enzyme is compatible with our XAS data. Cyanide (CN-) inactivated the enzyme by replacing the sulfido ligand at Mo(VI) with an oxo ligand. Evidence that the sulfido group may become protonated upon molybdenum reduction was obtained. Our results emphasize the role of coordination flexibility at the molybdenum center during inhibitory and catalytic processes of FDH enzymes.

The application of differential scanning fluorimetry in exploring bisubstrate binding to protein arginine N-methyltransferase 1.

Protein arginine N-methyltransferases (PRMTs) are a family of 9 enzymes that catalyze mono- or di-methylation of arginine residues using S-adenosyl-l-methionine (SAM). Arginine methylation is an important post-translational modification that can regulate the activity and structure of target proteins. Altered PRMT activity can lead to a variety of health issues including neurodevelopmental disease, autoimmune disorders, cancer, and cardiovascular disease. Thus, developing a robust mechanistic understanding of PRMT function may provide insight into these various disease states and enable the development of potential therapeutic agents. Although PRMTs have been studied for nearly two decades, a consensus regarding the mechanism of action for this class of enzymes has remained noticeably elusive. To address this shortcoming, differential scanning fluorimetry (DSF) was used to gain mechanistic insight into the order of PRMT substrate and cofactor binding. This methodology confirms that PRMT cofactor binding precedes target substrate binding and supports the use of DSF to study bisubstrate enzymatic reaction mechanisms.

Catalytic Mechanism of Aryl-Ether Bond Cleavage in Lignin by LigF and LigG.

Given the abundance of lignin in nature, multiple enzyme systems have been discovered to cleave the ß-O-4 bonds, the most prevalent intermonomer linkage. In particular, stereospecific cleavage of lignin oligomers by glutathione S-transferases (GSTs) has been reported in several sphingomonads. Here, we apply quantum mechanics/molecular mechanics simulations to study the mechanism of two glutathione-dependent enzymes in the ß-aryl ether catabolic pathway of Sphingomonas sp. SYK-6, namely, LigF, a ß-etherase, and LigG, a lyase. For LigF, the free-energy landscape supports a SN2 reaction mechanism, with the monoaromatic leaving group being promptly neutralized upon release. Specific interactions with conserved residues are responsible for stereoselectivity and for activation of the cofactor as a nucleophile. A glutathione conjugate is also released by LigF and serves the substrate of LigG, undergoing a SN2-like reaction, in which Cys15 acts as the nucleophile, to yield the second monoaromatic product. The simulations suggest that the electron-donating substituent at the para-position found in lignin-derived aromatics and the interaction with Tyr217 are essential for reactivity in LigG. Overall, this work deepens the understanding of the stereospecific enzymatic mechanisms in the ß-aryl ether cleavage pathway and reveals key structural features underpinning the ligninolytic activity detected in several sphingomonad GSTs.

Kinetic and structural evidence that Asp-678 plays multiple roles in catalysis by the quinoprotein glycine oxidase.

PlGoxA from Pseudoalteromonas luteoviolacea is a glycine oxidase that utilizes a protein-derived cysteine tryptophylquinone (CTQ) cofactor. A notable feature of its catalytic mechanism is that it forms a stable product-reduced CTQ adduct that is not hydrolyzed in the absence of O2 Asp-678 resides near the quinone moiety of PlGoxA, and an Asp is structurally conserved in this position in all tryptophylquinone enzymes. In those other enzymes, mutation of that Asp results in no or negligible CTQ formation. In this study, mutation of Asp-678 in PlGoxA did not abolish CTQ formation. This allowed, for the first time, studying the role of this residue in catalysis. D678A and D678N substitutions yielded enzyme variants with CTQ, which did not react with glycine, although glycine was present in the crystal structures in the active site. D678E PlGoxA was active but exhibited a much slower kcat This mutation altered the kinetic mechanism of the reductive half-reaction such that one could observe a previously undetected reactive intermediate, an initial substrate-oxidized CTQ adduct, which converted to the product-reduced CTQ adduct. These results indicate that Asp-678 is involved in the initial deprotonation of the amino group of glycine, enabling nucleophilic attack of CTQ, as well as the deprotonation of the substrate-oxidized CTQ adduct, which is coupled to CTQ reduction. The structures also suggest that Asp-678 is acting as a proton relay that directs these protons to a water channel that connects the active sites on the subunits of this homotetrameric enzyme.