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A major challenge in enzymology is the need to correlate the dynamic properties of enzymes with, and understand the impact on, their catalytic cycles. This is especially the case with large, multicenter enzymes such as the nitric oxide synthases (NOSs), where the importance of dynamics has been inferred from a variety of structural, single-molecule, and ensemble spectroscopic approaches but where motions have not been correlated experimentally with mechanistic steps in the reaction cycle. Here we take such an approach. Using time-resolved spectroscopy employing absorbance and Förster resonance energy transfer (FRET) and exploiting the properties of a flavin analogue (5-deazaflavin mononucleotide (5-dFMN)) and isotopically labeled nicotinamide coenzymes, we correlate the timing of CaM structural changes when bound to neuronal nitric oxide synthase (nNOS) with the nNOS catalytic cycle. We show that remodeling of CaM occurs early in the electron transfer sequence (FAD reduction), not at later points in the reaction cycle (e.g., FMN reduction). Conformational changes are tightly correlated with FAD reduction kinetics and reflect a transient "opening" and then "closure" of the bound CaM molecule. We infer that displacement of the C-terminal tail on binding NADPH and subsequent FAD reduction are the likely triggers of conformational change. By combining the use of cofactor/coenzyme analogues and time-resolved FRET/absorbance spectrophotometry, we show how the reaction cycles of complex enzymes can be simplified, enabling a detailed study of the relationship between protein dynamics and reaction cycle chemistry-an approach that can also be used with other complex multicenter enzymes.

The general requirement for conformational sampling in biological electron transfer reactions catalysed by multi-domain redox systems has been emphasized in recent years. Crucially, we lack insight into the extent of the conformational space explored and the nature of the energy landscapes associated with these reactions. The nitric oxide synthases (NOS) produce the signalling molecule NO through a series of complex electron transfer reactions. There is accumulating evidence that protein domain dynamics and calmodulin binding are implicated in regulating electron flow from NADPH, through the FAD and FMN cofactors, to the haem oxygenase domain, where NO is generated. Simple models based on static crystal structures of the isolated reductase domain have suggested a role for large-scale motions of the FMN-binding domain in shuttling electrons from the reductase domain to the oxygenase domain. However, detailed insight into the higher-order domain architecture and dynamic structural transitions in NOS enzymes during enzyme turnover is lacking. In this review, we discuss the recent advances made towards mapping the catalytic free energy landscapes of NOS enzymes through integration of both structural techniques (e.g. cryo-electron microscopy) and biophysical techniques (e.g. pulsed-electron paramagnetic resonance). The general picture that emerges from these experiments is that NOS enzymes exist in an equilibrium of conformations, comprising a 'rugged' or 'frustrated' energy landscape, with a key regulatory role for calmodulin in driving vectorial electron transfer by altering the conformational equilibrium. A detailed understanding of these landscapes may provide new opportunities for discovery of isoform-specific inhibitors that bind at the dynamic interfaces of these multi-dimensional energy landscapes., (© 2014 FEBS.)

Enzyme mechanisms are often probed by structure-informed point mutations and measurement of their effects on enzymatic properties to test mechanistic hypotheses. In many cases, the challenge is to report on complex, often inter-linked elements of catalysis. Evidence for long-range effects on enzyme mechanism resulting from mutations remains sparse, limiting the design/redesign of synthetic catalysts in a predictable way. Here we show that improving the accessibility of the active site pocket of copper nitrite reductase by mutation of a surface-exposed phenylalanine residue (Phe306), located 12 Å away from the catalytic site type-2 Cu (T2Cu), profoundly affects intra-molecular electron transfer, substrate-binding and catalytic activity. Structures and kinetic studies provide an explanation for the lower affinity for the substrate and the alteration of the rate-limiting step in the reaction. Our results demonstrate that distant residues remote from the active site can have marked effects on enzyme catalysis, by driving mechanistic change through relatively minor structural perturbations.

Vitamin C is a widely used vitamin. Here we review the occurrence and properties of aldonolactone oxidoreductases, an important group of flavoenzymes responsible for the ultimate production of vitamin C and its analogs in animals, plants, and single-cell organisms.

Unlabelled: l-galactono-1,4-lactone dehydrogenase (GALDH) catalyzes the terminal step of vitamin C biosynthesis in plant mitochondria. Here we investigated the communication between Arabidopsis thaliana GALDH and its natural electron acceptor cytochrome c (Cc). Using laser-generated radicals we observed the formation and stabilization of the GALDH semiquinone anionic species (GALDHSQ ). GALDHSQ oxidation by Cc exhibited a nonlinear dependence on Cc concentration consistent with a kinetic mechanism involving protein-partner association to form a transient bimolecular complex prior to the electron transfer step. Oxidation of GALDHSQ by Cc was significantly impaired at high ionic strength, revealing the existence of attractive charge-charge interactions between the two reactants. Isothermal titration calorimetry showed that GALDH weakly interacts with both oxidized and reduced Cc. Chemical shift perturbations for (1) H and (15) N nuclei of Cc, arising from the interactions with unlabeled GALDH, were used to map the interacting surface of Cc. For Arabidopsis Cc and yeast Cc, similar residues are involved in the interaction with GALDH. These residues are confined to a single surface surrounding the heme edge. The range of chemical shift perturbations for the physiological Arabidopsis Cc-GALDH complex is larger than that of the non-physiological yeast Cc-GALDH complex, indicating that the former complex is more specific. In summary, the results point to a relatively low affinity GALDH-Cc interaction, similar for all partner redox states, involving protein-protein dynamic motions. Evidence is also provided that Cc utilizes a conserved surface surrounding the heme edge for the interaction with GALDH and other redox partners., Database: NMR assignment of the backbone amide resonances of Arabidopsis CcRED has been deposited in BMRB database (BMRB accession number 18828). L-galactono-1,4-lactone dehydrogenase (L-galactono-1,4-lactone: ferricytochrome c oxidoreductase, EC 1.3.2.3)., (© 2013 The Authors Journal compilation © 2013 FEBS.)

Enzyme-catalysed electron transfer reactions are often controlled by protein motions and coupled to chemical change such as proton transfer. We have investigated the nature of this control in the blue copper-dependent nitrite reductase from Alcaligenes xylosoxidans (AxNiR). Inter-Cu electron transfer from the T1Cu site to the T2Cu catalytic site in AxNiR occurs via a proton-coupled electron transfer mechanism. Here we have studied the kinetics of both electron and proton transfer independently using laser-flash photolysis for native AxNiR and its proton-channel mutant N90S. In native AxNiR, both inter-Cu electron transfer and proton transfer exhibit similar rates, and show an unusual dependence on the nitrite concentration. An initial decrease in the observed rates at low nitrite concentrations is followed by an increase in the observed rates at high nitrite concentrations (> 5 mm). In N90S, in which the T1Cu reduction potential is elevated by 60 mV, no inter-Cu electron transfer or proton transfer was observed in the absence of nitrite. Only in the presence of nitrite were both processes detected, with similar [nitrite] dependence, but the nitrite dependence was different compared with native enzyme. The substrate dependence in N90S was similar to that observed in steady-state assays, suggesting that this substitution resulted in proton-coupled electron transfer becoming rate-limiting. A pH perturbation experiment with native AxNiR revealed that protonation triggers inter-Cu electron transfer and generation of NO. Our results show a strong coupling of inter-Cu electron transfer and proton transfer for both native AxNiR and N90S, and provide novel insights into the controlled delivery of electrons and protons to the substrate-utilization T2Cu active site of AxNiR., (© 2012 The Authors Journal compilation © 2012 FEBS.)

Biological electron transfer is a fundamentally important reaction. Despite the apparent simplicity of these reactions (in that no bonds are made or broken), their experimental interrogation is often complicated because of adiabatic control exerted through associated chemical and conformational change. We have studied the nature of this control in several enzyme systems, cytochrome P450 reductase, methionine synthase reductase and copper-dependent nitrite reductase. Specifically, we review the evidence for conformational control in cytochrome P450 reductase and methionine synthase reductase and chemical control i.e. proton coupled electron transfer in nitrite reductase. This evidence has accrued through the use and integration of structural, spectroscopic and advanced kinetic methods. This integrated approach is shown to be powerful in dissecting control mechanisms for biological electron transfer and will likely find widespread application in the study of related biological redox systems., (Copyright © 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

We demonstrated recently that two protons are involved in reduction of nitrite to nitric oxide through a proton-coupled electron transfer (ET) reaction catalyzed by the blue Cu-dependent nitrite reductase (Cu NiR) of Alcaligenes xylosoxidans (AxNiR). Here, the functionality of two putative proton channels, one involving Asn90 and the other His254, is studied using single (N90S, H254F) and double (N90S--H254F) mutants. All mutants studied are active, indicating that protons are still able to reach the active site. The H254F mutation has no effect on the catalytic activity, while the N90S mutation results in ~70% decrease in activity. Laser flash-photolysis experiments show that in H254F and wild-type enzyme electrons enter at the level of the T1Cu and then redistribute between the two Cu sites. Complete ET from T1Cu to T2Cu occurs only when nitrite binds at the T2Cu site. This indicates that substrate binding to T2Cu promotes ET from T1Cu, suggesting that the enzyme operates an ordered mechanism. In fact, in the N90S and N90S--H254F variants, where the T1Cu site redox potential is elevated by ∼60 mV, inter-Cu ET is only observed in the presence of nitrite. From these results it is evident that the Asn90 channel is the main proton channel in AxNiR, though protons can still reach the active site if this channel is disrupted. Crystallographic structures provide a clear structural rationale for these observations, including restoration of the proton delivery via a significant movement of the loop connecting the T1Cu ligands Cys130 and His139 that occurs on binding of nitrite. Notably, a role for this loop in facilitating interaction of cytochrome c(551) with Cu NiR has been suggested previously based on a crystal structure of the binary complex.

Trypanosoma cruzi, the aetiological agent of Chagas' disease, is unable to salvage vitamin C (l-ascorbate) from its environment and relies on de novo synthesis for its survival. Because humans lack the capacity to synthesize ascorbate, the trypanosomal enzymes involved in ascorbate biosynthesis are interesting targets for drug therapy. The terminal step in ascorbate biosynthesis is catalyzed by flavin-dependent aldonolactone oxidoreductases belonging to the vanillyl-alcohol oxidase (VAO) protein family. Here we studied the properties of recombinant T. cruzi galactonolactone oxidoreductase (TcGAL), refolded from inclusion bodies using a reverse micelles system. The refolded enzyme shows native-like secondary structure and is active with both l-galactono-1,4-lactone and d-arabinono-1,4-lactone. At odd with an earlier claim, TcGAL employs a non-covalently bound FAD as redox-active cofactor. Moreover, it is shown for the first time that TcGAL can use molecular oxygen as electron acceptor. This is in line with the absence of a recently identified gatekeeper residue that prevents aldonolactone oxidoreductases from plants to act as oxidases., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Ten years of experience with molecular class-specific information systems (MCSIS) such as with the hand-curated G protein-coupled receptor database (GPCRDB) or the semiautomatically generated nuclear receptor database has made clear that a wide variety of questions can be answered when protein-related data from many different origins can be flexibly combined. MCSISes revolve around a multiple sequence alignment (MSA) that includes "all" available sequences from the entire superfamily, and it has been shown at many occasions that the quality of these alignments is the most crucial aspect of the MCSIS approach. We describe here a system called 3DM that can automatically build an entire MCSIS. 3DM bases the MSA on a multiple structure alignment, which implies that the availability of a large number of superfamily members with a known three-dimensional structure is a requirement for 3DM to succeed well. Thirteen MCSISes were constructed and placed on the Internet for examination. These systems have been instrumental in a large series of research projects related to enzyme activity or the understanding and engineering of specificity, protein stability engineering, DNA-diagnostics, drug design, and so forth.

The flavoenzyme L-galactono-gamma-lactone dehydrogenase (GALDH) catalyzes the terminal step of vitamin C biosynthesis in plants. Little is known about the catalytic mechanism of GALDH and related aldonolactone oxidoreductases. Here we identified an essential Glu-Arg pair in the active site of GALDH from Arabidopsis thaliana. Glu386 and Arg388 variants show high K(m) values for L-galactono-1,4-lactone and low turnover rates. Arg388 is crucial for the stabilization of the anionic form of the reduced FAD cofactor. Glu386 is involved in productive substrate binding. The E386D variant has lost its specificity for L-galactono-1,4-lactone and shows the highest catalytic efficiency with L-gulono-1,4-lactone.

Correlated mutation analyses (CMA) on multiple sequence alignments are widely used for the prediction of the function of amino acids. The accuracy of CMA-based predictions is mainly determined by the number of sequences, by their evolutionary distances, and by the quality of the alignments. These criteria are best met in structure-based sequence alignments of large super-families. So far, CMA-techniques have mainly been employed to study the receptor interactions. The present work shows how a novel CMA tool, called Comulator, can be used to determine networks of functionally related residues in enzymes. These analyses provide leads for protein engineering studies that are directed towards modification of enzyme specificity or activity. As proof of concept, Comulator has been applied to four enzyme super-families: the isocitrate lyase/phoshoenol-pyruvate mutase super-family, the hexokinase super-family, the RmlC-like cupin super-family, and the FAD-linked oxidases super-family. In each of those cases networks of functionally related residue positions were discovered that upon mutation influenced enzyme specificity and/or activity as predicted. We conclude that CMA is a powerful tool for redesigning enzyme activity and selectivity., (2009 Wiley-Liss, Inc.)

The mitochondrial flavoenzyme l-galactono-gamma-lactone dehydrogenase (GALDH) catalyzes the ultimate step of vitamin C biosynthesis in plants. We found that recombinant GALDH from Arabidopsis (Arabidopsis thaliana) is inactivated by hydrogen peroxide due to selective oxidation of cysteine (Cys)-340, located in the cap domain. Electrospray ionization mass spectrometry revealed that the partial reversible oxidative modification of Cys-340 involves the sequential formation of sulfenic, sulfinic, and sulfonic acid states. S-Glutathionylation of the sulfenic acid switches off GALDH activity and protects the enzyme against oxidative damage in vitro. C340A and C340S GALDH variants are insensitive toward thiol oxidation, but exhibit a poor affinity for l-galactono-1,4-lactone. Cys-340 is buried beneath the protein surface and its estimated pK(a) of 6.5 suggests the involvement of the thiolate anion in substrate recognition. The indispensability of a redox-sensitive thiol provides a rationale why GALDH was designed as a dehydrogenase and not, like related aldonolactone oxidoreductases, as an oxidase.

The oxygen reactivity of flavoproteins is poorly understood. Here we show that a single Ala to Gly substitution in l-galactono-gamma-lactone dehydrogenase (GALDH) turns the enzyme into a catalytically competent oxidase. GALDH is an aldonolactone oxidoreductase with a vanillyl-alcohol oxidase (VAO) fold. We found that nearly all oxidases in the VAO family contain either a Gly or a Pro at a structurally conserved position near the C4a locus of the isoalloxazine moiety of the flavin, whereas dehydrogenases prefer another residue at this position. Mutation of the corresponding residue in GALDH (Ala-113 --> Gly) resulted in a striking 400-fold increase in oxygen reactivity, whereas the cytochrome c reductase activity is retained. The activity of the A113G variant shows a linear dependence on oxygen concentration (k(ox) = 3.5 x 10(5) m(-1) s(-1)), similar to most other flavoprotein oxidases. The Ala-113 --> Gly replacement does not change the reduction potential of the flavin but creates space for molecular oxygen to react with the reduced flavin. In the wild-type enzyme, Ala-113 acts as a gatekeeper, preventing oxygen from accessing the isoalloxazine nucleus. The presence of such an oxygen access gate seems to be a key factor for the prevention of oxidase activity within the VAO family and is absent in members that act as oxidases.

There is considerable interest in the use of enantioselective alcohol dehydrogenases for the production of enantio- and diastereomerically pure diols, which are important building blocks for pharmaceuticals, agrochemicals and fine chemicals. Due to the need for a stable alcohol dehydrogenase with activity at low-temperature process conditions (30 degrees C) for the production of (2S,5S)-hexanediol, we have improved an alcohol dehydrogenase from the hyperthermophilic archaeon Pyrococcus furiosus (AdhA). A stable S-selective alcohol dehydrogenase with increased activity at 30 degrees C on the substrate 2,5-hexanedione was generated by laboratory evolution on the thermostable alcohol dehydrogenase AdhA. One round of error-prone PCR and screening of approximately 1,500 mutants was performed. The maximum specific activity of the best performing mutant with 2,5-hexanedione at 30 degrees C was tenfold higher compared to the activity of the wild-type enzyme. A 3D-model of AdhA revealed that this mutant has one mutation in the well-conserved NADP(H)-binding site (R11L), and a second mutation (A180V) near the catalytic and highly conserved threonine at position 183.

The VAO flavoprotein family is a rapidly growing family of oxidoreductases that favor the covalent binding of the FAD cofactor. In this review we report on the catalytic properties of some newly discovered VAO family members and their mode of flavin binding. Covalent binding of the flavin is a self-catalytic post-translational modification primarily taking place in oxidases. Covalent flavinylation increases the redox potential of the cofactor and thus its oxidation power. Recent findings have revealed that some members of the VAO family anchor the flavin via a dual covalent linkage (6-S-cysteinyl-8alpha-N1-histidyl FAD). Some VAO-type aldonolactone oxidoreductases favor the non-covalent binding of the flavin cofactor. These enzymes act as dehydrogenases, using cytochrome c as electron acceptor.

l-Galactono-1,4-lactone dehydrogenase (GALDH; ferricytochrome c oxidoreductase; EC 1.3.2.3) is a mitochondrial flavoenzyme that catalyzes the final step in the biosynthesis of vitamin C (l-ascorbic acid) in plants. In the present study, we report on the biochemical properties of recombinant Arabidopsis thaliana GALDH (AtGALDH). AtGALDH oxidizes, in addition to l-galactono-1,4-lactone (K(m) = 0.17 mm, k(cat) = 134 s(-1)), l-gulono-1,4-lactone (K(m) = 13.1 mm, k(cat) = 4.0 s(-1)) using cytochrome c as an electron acceptor. Aerobic reduction of AtGALDH with the lactone substrate generates the flavin hydroquinone. The two-electron reduced enzyme reacts poorly with molecular oxygen (k(ox) = 6 x 10(2) m(-1).s(-1)). Unlike most flavoprotein dehydrogenases, AtGALDH forms a flavin N5 sulfite adduct. Anaerobic photoreduction involves the transient stabilization of the anionic flavin semiquinone. Most aldonolactone oxidoreductases contain a histidyl-FAD as a covalently bound prosthetic group. AtGALDH lacks the histidine involved in covalent FAD binding, but contains a leucine instead (Leu56). Leu56 replacements did not result in covalent flavinylation but revealed the importance of Leu56 for both FAD-binding and catalysis. The Leu56 variants showed remarkable differences in Michaelis constants for both l-galactono-1,4-lactone and l-gulono-1,4-lactone and released their FAD cofactor more easily than wild-type AtGALDH. The present study provides the first biochemical characterization of AtGALDH and some active site variants. The role of GALDH and the possible involvement of other aldonolactone oxidoreductases in the biosynthesis of vitamin C in A. thaliana are also discussed.