Publications by authors named "Simon de Vries"

47 Publications

Microsecond time-scale kinetics of transient biochemical reactions.

PLoS One 2017 3;12(10):e0185888. Epub 2017 Oct 3.

Department of Biotechnology, Delft University of Technology, Delft, The Netherlands.

To afford mechanistic studies in enzyme kinetics and protein folding in the microsecond time domain we have developed a continuous-flow microsecond time-scale mixing instrument with an unprecedented dead-time of 3.8 ± 0.3 μs. The instrument employs a micro-mixer with a mixing time of 2.7 μs integrated with a 30 mm long flow-cell of 109 μm optical path length constructed from two parallel sheets of silver foil; it produces ultraviolet-visible spectra that are linear in absorbance up to 3.5 with a spectral resolution of 0.4 nm. Each spectrum corresponds to a different reaction time determined by the distance from the mixer outlet, and by the fluid flow rate. The reaction progress is monitored in steps of 0.35 μs for a total duration of ~600 μs. As a proof of principle the instrument was used to study spontaneous protein refolding of pH-denatured cytochrome c. Three folding intermediates were determined: after a novel, extremely rapid initial phase with τ = 4.7 μs, presumably reflecting histidine re-binding to the iron, refolding proceeds with time constants of 83 μs and 345 μs to a coordinatively saturated low-spin iron form in quasi steady state. The time-resolution specifications of our spectrometer for the first time open up the general possibility for comparison of real data and molecular dynamics calculations of biomacromolecules on overlapping time scales.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0185888PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5626514PMC
October 2017

Affordances and neuroscience: Steps towards a successful marriage.

Neurosci Biobehav Rev 2017 Sep 27;80:622-629. Epub 2017 Jul 27.

Center for Human Movement Sciences, University of Groningen, University Medical Center Groningen, The Netherlands.

The concept of affordance is rapidly gaining popularity in neuroscientific accounts of perception and action. This concept was introduced by James Gibson to refer to the action possibilities of the environment. By contrast, standard cognitive neuroscience typically uses the concept to refer to (action-oriented) representations in the brain. This paper will show that the view of affordances as representations firmly places the concept in the subject-object framework that dominates both psychology and neuroscience. Notably, Gibson introduced the affordance concept to overcome this very framework. We describe an account of the role of the brain in perception and action that is consistent with Gibson. Making use of neuroscientific findings of neural reuse, degeneracy and functional connectivity, we conceptualize neural regions in the brain as dispositional parts of perceptual and action systems that temporarily assemble to enable animals to directly perceive and - in the paradigmatic case - utilize the affordances of the environment.
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http://dx.doi.org/10.1016/j.neubiorev.2017.07.008DOI Listing
September 2017

The cooperativity effect in the reaction of soluble quinoprotein (PQQ-containing) glucose dehydrogenase is not due to subunit interaction but to substrate-assisted catalysis.

FEBS J 2016 10;283(19):3604-3612

Department of Biotechnology, Delft University of Technology, The Netherlands.

Soluble quinoprotein (PQQ-containing) glucose dehydrogenase (sGDH, EC 1.1.99.35) catalyzes the oxidation of β-d-glucose to d-glucono-δ-lactone. Although sGDH has many analytical applications, the relationship between activity and substrate concentration is not well established. Previous steady-state kinetic studies revealed a negative cooperativity effect which has recently been ascribed to subunit interaction. To investigate this conclusion, stopped-flow kinetic experiments were carried out on the reaction in which oxidized enzyme (E ) was reduced with substrates to E . The appearance of E is observed to be preceded by formation of an intermediate enzyme form, Int, which is mono-exponentially formed from E . However, the rate of conversion of Int into E depends hyperbolically on the concentration of substrate (leading to a 35-fold stimulation in the case of glucose). Evidence is provided that substrate not only binds to E but also to Int and E as well, and that the binding to Int causes the significant stimulation of Int decay. It is proposed that a proton shuffling step is involved in the decay, which is facilitated by binding of substrate to Int. Substituting the PQQ-activating Ca by a Ba ion lowered all reaction rates but did not change the stimulation factor. In summary, the previous proposal that the cooperativity effect of sGDH is due to interaction between its substrate-loaded subunits is incorrect; it is due to substrate-assisted catalysis of the enzyme.

Enzymes: EC 1.1.99.35 - soluble quinoprotein glucose dehydrogenase.
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http://dx.doi.org/10.1111/febs.13829DOI Listing
October 2016

Characterization of Anammox Hydrazine Dehydrogenase, a Key N2-producing Enzyme in the Global Nitrogen Cycle.

J Biol Chem 2016 08 17;291(33):17077-92. Epub 2016 Jun 17.

From the Department of Microbiology, Institute for Water and Wetland Research, Radboud University Nijmegen, 6525 AJ Nijmegen, The Netherlands,

Anaerobic ammonium-oxidizing (anammox) bacteria derive their energy for growth from the oxidation of ammonium with nitrite as the electron acceptor. N2, the end product of this metabolism, is produced from the oxidation of the intermediate, hydrazine (N2H4). Previously, we identified N2-producing hydrazine dehydrogenase (KsHDH) from the anammox organism Kuenenia stuttgartiensis as the gene product of kustc0694 and determined some of its catalytic properties. In the genome of K. stuttgartiensis, kustc0694 is one of 10 paralogs related to octaheme hydroxylamine (NH2OH) oxidoreductase (HAO). Here, we characterized KsHDH as a covalently cross-linked homotrimeric octaheme protein as found for HAO and HAO-related hydroxylamine-oxidizing enzyme kustc1061 from K. stuttgartiensis Interestingly, the HDH trimers formed octamers in solution, each octamer harboring an amazing 192 c-type heme moieties. Whereas HAO and kustc1061 are capable of hydrazine oxidation as well, KsHDH was highly specific for this activity. To understand this specificity, we performed detailed amino acid sequence analyses and investigated the catalytic and spectroscopic (electronic absorbance, EPR) properties of KsHDH in comparison with the well defined HAO and kustc1061. We conclude that HDH specificity is most likely derived from structural changes around the catalytic heme 4 (P460) and of the electron-wiring circuit comprising seven His/His-ligated c-type hemes in each subunit. These nuances make HDH a globally prominent N2-producing enzyme, next to nitrous oxide (N2O) reductase from denitrifying microorganisms.
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http://dx.doi.org/10.1074/jbc.M116.735530DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5016112PMC
August 2016

Cytochrome bd Displays Significant Quinol Peroxidase Activity.

Sci Rep 2016 06 9;6:27631. Epub 2016 Jun 9.

Department of Biotechnology, Delft University of Technology, The Netherlands.

Cytochrome bd is a prokaryotic terminal oxidase that catalyses the electrogenic reduction of oxygen to water using ubiquinol as electron donor. Cytochrome bd is a tri-haem integral membrane enzyme carrying a low-spin haem b558, and two high-spin haems: b595 and d. Here we show that besides its oxidase activity, cytochrome bd from Escherichia coli is a genuine quinol peroxidase (QPO) that reduces hydrogen peroxide to water. The highly active and pure enzyme preparation used in this study did not display the catalase activity recently reported for E. coli cytochrome bd. To our knowledge, cytochrome bd is the first membrane-bound quinol peroxidase detected in E. coli. The observation that cytochrome bd is a quinol peroxidase, can provide a biochemical basis for its role in detoxification of hydrogen peroxide and may explain the frequent findings reported in the literature that indicate increased sensitivity to hydrogen peroxide and decreased virulence in mutants that lack the enzyme.
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http://dx.doi.org/10.1038/srep27631DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4899803PMC
June 2016

The multitude of iron-sulfur clusters in respiratory complex I.

Biochim Biophys Acta 2016 Aug 2;1857(8):1068-1072. Epub 2016 Mar 2.

Albert-Ludwigs-Universität Freiburg, Institut für Biochemie, Albertstr. 21, 79104 Freiburg i. Br., Germany. Electronic address:

Respiratory complex I couples the electron transfer from NADH to ubiquinone with the translocation of protons across the membrane. Complex I contains one non-covalently bound flavin mononucleotide and, depending on the species, up to ten iron-sulfur (Fe/S) clusters as cofactors. The reason for the presence of the multitude of Fe/S clusters in complex I remained enigmatic for a long time. The question was partly answered by investigations on the evolution of the complex revealing the stepwise construction of the electron transfer domain from several modules. Extension of the ancestral to the modern electron input domain was associated with the acquisition of several Fe/S-proteins. The X-ray structure of the complex showed that the NADH oxidation-site is connected with the quinone-reduction site by a chain of seven Fe/S-clusters. Fast enzyme kinetics revealed that this chain of Fe/S-clusters is used to regulate electron-tunneling rates within the complex. A possible function of the off-pathway cluster N1a is discussed. This article is part of a Special Issue entitled 'EBEC 2016: 19th European Bioenergetics Conference, Riva del Garda, Italy, July 2-6, 2016', edited by Prof. Paolo Bernardi.
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http://dx.doi.org/10.1016/j.bbabio.2016.02.018DOI Listing
August 2016

Better than Nature: Nicotinamide Biomimetics That Outperform Natural Coenzymes.

J Am Chem Soc 2016 Jan 13;138(3):1033-9. Epub 2016 Jan 13.

BBSRC/EPSRC Centre for Synthetic Biology of Fine and Speciality Chemicals, Faculty of Life Sciences, Manchester Institute of Biotechnology , 131 Princess Street, Manchester M1 7DN, United Kingdom.

The search for affordable, green biocatalytic processes is a challenge for chemicals manufacture. Redox biotransformations are potentially attractive, but they rely on unstable and expensive nicotinamide coenzymes that have prevented their widespread exploitation. Stoichiometric use of natural coenzymes is not viable economically, and the instability of these molecules hinders catalytic processes that employ coenzyme recycling. Here, we investigate the efficiency of man-made synthetic biomimetics of the natural coenzymes NAD(P)H in redox biocatalysis. Extensive studies with a range of oxidoreductases belonging to the "ene" reductase family show that these biomimetics are excellent analogues of the natural coenzymes, revealed also in crystal structures of the ene reductase XenA with selected biomimetics. In selected cases, these biomimetics outperform the natural coenzymes. "Better-than-Nature" biomimetics should find widespread application in fine and specialty chemicals production by harnessing the power of high stereo-, regio-, and chemoselective redox biocatalysts and enabling reactions under mild conditions at low cost.
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http://dx.doi.org/10.1021/jacs.5b12252DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4731831PMC
January 2016

Homeostatic control of nitric oxide (NO) at nanomolar concentrations in denitrifying bacteria - modelling and experimental determination of NO reductase kinetics in vivo in Paracoccus denitrificans.

Environ Microbiol 2016 09 25;18(9):2964-78. Epub 2016 Jan 25.

Department of Environmental Sciences, Norwegian University of Life Sciences, Ås, Norway.

Homeostatic control of nitric oxide (NO) at nanomolar concentrations appears common among denitrifying bacteria, often ascribed to synchronized expression of nitrite and nitric oxide reductase (Nir and Nor). We questioned whether this is sufficient: using the reported substrate affinities for cytochrome cd1 nitrite reductase (cNor), our model of batch cultures of Paracoccus denitrificans predicted NO concentrations orders of magnitude higher than measured. We rejected a hypothesis that the homeostatic control is due to a negative feedback by NO on the activity of NirS because the inclusion of such feedback resulted in too slow anaerobic growth and N2 production. We proceeded by determining the kinetic parameters for cNor in vivo by a carefully designed experiment, allowing the estimation of NO concentration at the cell surface while anoxic cultures depleted low headspace doses of NO. With the new parameters for cNor kinetics in vivo {v = vmax /[1 + K2 /(NO) + K1  × K2 /(NO)(2) ]; vmax  = 3.56 fmol NO cell(-1)  h(-1) , K1  < 1 nM, and K2  = 34 nM}, the model predicted NO concentrations close to that measured. Thus, enzyme kinetics alone can explain the observed NO homeostasis. Determinations of enzyme kinetic parameters in vivo are not trivial but evidently required to understand and model NO kinetics in denitrifying organisms in soils and aquatic environments.
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http://dx.doi.org/10.1111/1462-2920.13129DOI Listing
September 2016

The inner workings of the hydrazine synthase multiprotein complex.

Nature 2015 Nov 19;527(7578):394-7. Epub 2015 Oct 19.

Department of Biomolecular Mechanisms, Max Planck Institute for Medical Research, 69120 Heidelberg, Germany.

Anaerobic ammonium oxidation (anammox) has a major role in the Earth's nitrogen cycle and is used in energy-efficient wastewater treatment. This bacterial process combines nitrite and ammonium to form dinitrogen (N2) gas, and has been estimated to synthesize up to 50% of the dinitrogen gas emitted into our atmosphere from the oceans. Strikingly, the anammox process relies on the highly unusual, extremely reactive intermediate hydrazine, a compound also used as a rocket fuel because of its high reducing power. So far, the enzymatic mechanism by which hydrazine is synthesized is unknown. Here we report the 2.7 Å resolution crystal structure, as well as biophysical and spectroscopic studies, of a hydrazine synthase multiprotein complex isolated from the anammox organism Kuenenia stuttgartiensis. The structure shows an elongated dimer of heterotrimers, each of which has two unique c-type haem-containing active sites, as well as an interaction point for a redox partner. Furthermore, a system of tunnels connects these active sites. The crystal structure implies a two-step mechanism for hydrazine synthesis: a three-electron reduction of nitric oxide to hydroxylamine at the active site of the γ-subunit and its subsequent condensation with ammonia, yielding hydrazine in the active centre of the α-subunit. Our results provide the first, to our knowledge, detailed structural insight into the mechanism of biological hydrazine synthesis, which is of major significance for our understanding of the conversion of nitrogenous compounds in nature.
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http://dx.doi.org/10.1038/nature15517DOI Listing
November 2015

An electrogenic nitric oxide reductase.

FEBS Lett 2015 Jul 3;589(16):2050-7. Epub 2015 Jul 3.

Department of Biotechnology, Delft University of Technology, Julianalaan 67, 2628 BC, The Netherlands. Electronic address:

Nitric oxide reductases (Nors) are members of the heme-copper oxidase superfamily that reduce nitric oxide (NO) to nitrous oxide (N₂O). In contrast to the proton-pumping cytochrome oxidases, Nors studied so far have neither been implicated in proton pumping nor have they been experimentally established as electrogenic. The copper-A-dependent Nor from Bacillus azotoformans uses cytochrome c₅₅₁ as electron donor but lacks menaquinol activity, in contrast to our earlier report (Suharti et al., 2001). Employing reduced phenazine ethosulfate (PESH) as electron donor, the main NO reduction pathway catalyzed by Cu(A)Nor reconstituted in liposomes involves transmembrane cycling of the PES radical. We show that Cu(A)Nor reconstituted in liposomes generates a proton electrochemical gradient across the membrane similar in magnitude to cytochrome aa₃, highlighting that bacilli using Cu(A)Nor can exploit NO reduction for increased cellular ATP production compared to organisms using cNor.
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http://dx.doi.org/10.1016/j.febslet.2015.06.033DOI Listing
July 2015

The cytochrome ba3 oxidase from Thermus thermophilus does not generate a tryptophan radical during turnover: Implications for the mechanism of proton pumping.

Biochim Biophys Acta 2015 Oct 22;1847(10):1093-100. Epub 2015 May 22.

Laboratory of Biotechnology, Section Biocatalysis, Delft University of Technology, Julianalaan 67, 2628BC Delft, The Netherlands. Electronic address:

Oxygen reduction by cytochrome ba3 oxidase from Thermus thermophilus was studied by stopped-flow and microsecond freeze-hyperquenching analyzed with UV-Vis and EPR spectroscopy. In the initial phase, the low-spin heme b560 is rapidly and almost completely oxidized (kobs>33,000s(-1)) whereas CuA remains nearly fully reduced. The internal equilibrium between CuA and heme b560 with forward and reverse rate constants of 4621s(-1) and 3466s(-1), respectively, indicates a ~7.5mV lower midpoint potential for CuA compared to heme b560. The formation of the oxidized enzyme is relatively slow (693s(-1)). In contrast to the Paracoccus denitrificans cytochrome aa3 oxidase, where in the last phase of the oxidative half cycle a radical from the strictly conserved Trp272 is formed, no radical is formed in the cytochrome ba3 oxidase. Mutation of the Trp229, the cytochrome ba3 oxidase homologue to the Trp272, did not abolish the activity, again in contrast to the Paracoccus cytochrome aa3 oxidase. Differences in the proton pumping mechanisms of Type A and Type B oxidases are discussed in view of the proposed role of the strictly conserved tryptophan residue in the mechanism of redox-linked proton pumping in Type A oxidases. In spite of the differences between the Type A and Type B oxidases, we conclude that protonation of the proton-loading site constitutes the major rate-limiting step in both catalytic cycles.
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http://dx.doi.org/10.1016/j.bbabio.2015.05.013DOI Listing
October 2015

Transfer of attunement in length perception by dynamic touch.

Atten Percept Psychophys 2015 May;77(4):1396-410

Center for Human Movement Sciences, University Medical Center Groningen, University of Groningen, P.O. Box 196, 9700 AD, Groningen, The Netherlands.

Earlier studies have revealed that the calibration of an action sometimes transfers in a functionally specific way-the calibration of one action transfers to other actions that serve the same goal, even when they are performed with different anatomical structures. In the present study, we tested whether attunement (the process by which perceivers learn to detect a more useful, specifying, informational pattern) follows such a functional organization. Participants were trained to perceive the length of rods by dynamic touch with one of their effectors. It was found that training the right hand resulted in an attunement to a specifying variable with both hands, but not with the feet. Training the other limbs did not result in attunement. However, substantial individual differences were found. The implications of the results are explored for theories on the organization of perceptual learning and discussions on individual differences in perception.
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http://dx.doi.org/10.3758/s13414-015-0872-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4415978PMC
May 2015

Electron tunneling rates in respiratory complex I are tuned for efficient energy conversion.

Angew Chem Int Ed Engl 2015 Feb 19;54(9):2844-8. Epub 2015 Jan 19.

Department of Biotechnology, Institution Delft University of Technology, Julianalaan 67, 2628 BC, Delft (The Netherlands).

Respiratory complex I converts the free energy of ubiquinone reduction by NADH into a proton motive force, a redox reaction catalyzed by flavin mononucleotide(FMN) and a chain of seven iron-sulfur centers. Electron transfer rates between the centers were determined by ultrafast freeze-quenching and analysis by EPR and UV/Vis spectroscopy. The complex rapidly oxidizes three NADH molecules. The electron-tunneling rate between the most distant centers in the middle of the chain depends on the redox state of center N2 at the end of the chain, and is sixfold slower when N2 is reduced. The conformational changes that accompany reduction of N2 decrease the electronic coupling of the longest electron-tunneling step. The chain of iron-sulfur centers is not just a simple electron-conducting wire; it regulates the electron-tunneling rate synchronizing it with conformation-mediated proton pumping, enabling efficient energy conversion. Synchronization of rates is a principle means of enhancing the specificity of enzymatic reactions.
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http://dx.doi.org/10.1002/anie.201410967DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4506566PMC
February 2015

Design of turbulent tangential micro-mixers that mix liquids on the nanosecond time scale.

Anal Biochem 2015 Jan 16;469:19-26. Epub 2014 Oct 16.

Laboratory of Biotechnology, Delft University of Technology, 2628 BC Delft, The Netherlands. Electronic address:

Unravelling (bio)chemical reaction mechanisms and macromolecular folding pathways on the (sub)microsecond time scale is limited by the time resolution of kinetic instruments for mixing reactants and observation of the progress of the reaction. To improve the mixing time resolution, turbulent four- and two-jet tangential micro-mixers were designed and characterized for their mixing and (unwanted) premixing performances employing acid-base reactions monitored by a pH-sensitive fluorescent dye. The mixing performances of the micro-mixers were determined after the mixing chamber in a free-flowing jet. The premixing behavior in the vortex chamber was assessed in an optically transparent glass-silicon replica of a previously well-characterized stainless-steel four-jet tangential micro-mixer. At the highest flow rates, complete mixing was achieved in 160ns with only approximately 9% premixing of the reactants. The mixing time of 160ns is at least 50 times shorter than estimated for other fast mixing devices. Key aspects to the design of ultrafast turbulent micro-mixers are discussed. The integration of these micro-mixers with an optical flow cell would enable the study of the very onset of chemical reactions in general and of enzyme catalytic reactions in particular.
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http://dx.doi.org/10.1016/j.ab.2014.10.003DOI Listing
January 2015

Dancers entrain more effectively than non-dancers to another actor's movements.

Front Hum Neurosci 2014 8;8:800. Epub 2014 Oct 8.

Department of Psychology, Center for Cognition, Action and Perception, University of Cincinnati Cincinnati, OH, USA.

For many everyday sensorimotor tasks, trained dancers have been found to exhibit distinct and sometimes superior (more stable or robust) patterns of behavior compared to non-dancers. Past research has demonstrated that experts in fields requiring specialized physical training and behavioral control exhibit superior interpersonal coordination capabilities for expertise-related tasks. To date, however, no published studies have compared dancers' abilities to coordinate their movements with the movements of another individual-i.e., during a so-called visual-motor interpersonal coordination task. The current study was designed to investigate whether trained dancers would be better able to coordinate with a partner performing short sequences of dance-like movements than non-dancers. Movement time series were recorded for individual dancers and non-dancers asked to synchronize with a confederate during three different movement sequences characterized by distinct dance styles (i.e., dance team routine, contemporary ballet, mixed style) without hearing any auditory signals or music. A diverse range of linear and non-linear analyses (i.e., cross-correlation, cross-recurrence quantification analysis, and cross-wavelet analysis) provided converging measures of coordination across multiple time scales. While overall levels of interpersonal coordination were influenced by differences in movement sequence for both groups, dancers consistently displayed higher levels of coordination with the confederate at both short and long time scales. These findings demonstrate that the visual-motor coordination capabilities of trained dancers allow them to better synchronize with other individuals performing dance-like movements than non-dancers. Further investigation of similar tasks may help to increase the understanding of visual-motor entrainment in general, as well as provide insight into the effects of focused training on visual-motor and interpersonal coordination.
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http://dx.doi.org/10.3389/fnhum.2014.00800DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4189607PMC
October 2014

Engineering acetyl coenzyme A supply: functional expression of a bacterial pyruvate dehydrogenase complex in the cytosol of Saccharomyces cerevisiae.

mBio 2014 Oct 21;5(5):e01696-14. Epub 2014 Oct 21.

Department of Biotechnology, Delft University of Technology, Delft, The Netherlands

The energetic (ATP) cost of biochemical pathways critically determines the maximum yield of metabolites of vital or commercial relevance. Cytosolic acetyl coenzyme A (acetyl-CoA) is a key precursor for biosynthesis in eukaryotes and for many industrially relevant product pathways that have been introduced into Saccharomyces cerevisiae, such as isoprenoids or lipids. In this yeast, synthesis of cytosolic acetyl-CoA via acetyl-CoA synthetase (ACS) involves hydrolysis of ATP to AMP and pyrophosphate. Here, we demonstrate that expression and assembly in the yeast cytosol of an ATP-independent pyruvate dehydrogenase complex (PDH) from Enterococcus faecalis can fully replace the ACS-dependent pathway for cytosolic acetyl-CoA synthesis. In vivo activity of E. faecalis PDH required simultaneous expression of E. faecalis genes encoding its E1α, E1β, E2, and E3 subunits, as well as genes involved in lipoylation of E2, and addition of lipoate to growth media. A strain lacking ACS that expressed these E. faecalis genes grew at near-wild-type rates on glucose synthetic medium supplemented with lipoate, under aerobic and anaerobic conditions. A physiological comparison of the engineered strain and an isogenic Acs(+) reference strain showed small differences in biomass yields and metabolic fluxes. Cellular fractionation and gel filtration studies revealed that the E. faecalis PDH subunits were assembled in the yeast cytosol, with a subunit ratio and enzyme activity similar to values reported for PDH purified from E. faecalis. This study indicates that cytosolic expression and assembly of PDH in eukaryotic industrial microorganisms is a promising option for minimizing the energy costs of precursor supply in acetyl-CoA-dependent product pathways. Importance: Genetically engineered microorganisms are intensively investigated and applied for production of biofuels and chemicals from renewable sugars. To make such processes economically and environmentally sustainable, the energy (ATP) costs for product formation from sugar must be minimized. Here, we focus on an important ATP-requiring process in baker's yeast (Saccharomyces cerevisiae): synthesis of cytosolic acetyl coenzyme A, a key precursor for many industrially important products, ranging from biofuels to fragrances. We demonstrate that pyruvate dehydrogenase from the bacterium Enterococcus faecalis, a huge enzyme complex with a size similar to that of a ribosome, can be functionally expressed and assembled in the cytosol of baker's yeast. Moreover, we show that this ATP-independent mechanism for cytosolic acetyl-CoA synthesis can entirely replace the ATP-costly native yeast pathway. This work provides metabolic engineers with a new option to optimize the performance of baker's yeast as a "cell factory" for sustainable production of fuels and chemicals.
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http://dx.doi.org/10.1128/mBio.01696-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4212835PMC
October 2014

Characterization of the RnfB and RnfG subunits of the Rnf complex from the archaeon Methanosarcina acetivorans.

PLoS One 2014 16;9(5):e97966. Epub 2014 May 16.

Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania, United States of America.

Rnf complexes are redox-driven ion pumps identified in diverse species from the domains Bacteria and Archaea, biochemical characterizations of which are reported for two species from the domain Bacteria. Here, we present characterizations of the redox-active subunits RnfG and RnfB from the Rnf complex of Methanosarcina acetivorans, an acetate-utilizing methane-producing species from the domain Archaea. The purified RnfG subunit produced in Escherichia coli fluoresced in SDS-PAGE gels under UV illumination and showed a UV-visible spectrum typical of flavoproteins. The Thr166Gly variant of RnfG was colorless and failed to fluoresce under UV illumination confirming a role for Thr166 in binding FMN. Redox titration of holo-RnfG revealed a midpoint potential of -129 mV for FMN with n = 2. The overproduced RnfG was primarily localized to the membrane of E. coli and the sequence contained a transmembrane helix. A topological analysis combining reporter protein fusion and computer predictions indicated that the C-terminal domain containing FMN is located on the outer aspect of the cytoplasmic membrane. The purified RnfB subunit produced in E. coli showed a UV-visible spectrum typical of iron-sulfur proteins. The EPR spectra of reduced RnfB featured a broad spectral shape with g values (2.06, 1.94, 1.90, 1.88) characteristic of magnetically coupled 3Fe-4S and 4Fe-4S clusters in close agreement with the iron and acid-labile sulfur content. The ferredoxin specific to the aceticlastic pathway served as an electron donor to RnfB suggesting this subunit is the entry point of electrons to the Rnf complex. The results advance an understanding of the organization and biochemical properties of the Rnf complex and lay a foundation for further understanding the overall mechanism in the pathway of methane formation from acetate.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0097966PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4023990PMC
August 2015

Metabolic modeling of denitrification in Agrobacterium tumefaciens: a tool to study inhibiting and activating compounds for the denitrification pathway.

Front Microbiol 2012 18;3:370. Epub 2012 Oct 18.

Department of Biotechnology, Delft University of Technology Delft, Netherlands.

A metabolic network model for facultative denitrification was developed based on experimental data obtained with Agrobacterium tumefaciens. The model includes kinetic regulation at the enzyme level and transcription regulation at the enzyme synthesis level. The objective of this work was to study the key factors regulating the metabolic response of the denitrification pathway to transition from oxic to anoxic respiration and to find parameter values for the biological processes that were modeled. The metabolic model was used to test hypotheses that were formulated based on the experimental results and offers a structured look on the processes that occur in the cell during transition in respiration. The main phenomena that were modeled are the inhibition of the cytochrome c oxidase by nitric oxide (NO) and the (indirect) inhibition of oxygen on the denitrification enzymes. The activation of transcription of nitrite reductase and NO reductase by their respective substrates were hypothesized. The general assumption that nitrite and NO reduction are controlled interdependently to prevent NO accumulation does not hold for A. tumefaciens. The metabolic network model was demonstrated to be a useful tool for unraveling the different factors involved in the complex response of A. tumefaciens to highly dynamic environmental conditions.
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http://dx.doi.org/10.3389/fmicb.2012.00370DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3475394PMC
October 2012

Oxoferryl-porphyrin radical catalytic intermediate in cytochrome bd oxidases protects cells from formation of reactive oxygen species.

J Biol Chem 2012 Mar 27;287(12):8830-8. Epub 2012 Jan 27.

Department of Biotechnology, Section Enzymology, Delft University of Technology, Delft, The Netherlands.

The quinol-linked cytochrome bd oxidases are terminal oxidases in respiration. These oxidases harbor a low spin heme b(558) that donates electrons to a binuclear heme b(595)/heme d center. The reaction with O(2) and subsequent catalytic steps of the Escherichia coli cytochrome bd-I oxidase were investigated by means of ultra-fast freeze-quench trapping followed by EPR and UV-visible spectroscopy. After the initial binding of O(2), the O-O bond is heterolytically cleaved to yield a kinetically competent heme d oxoferryl porphyrin π-cation radical intermediate (compound I) magnetically interacting with heme b(595). Compound I accumulates to 0.75-0.85 per enzyme in agreement with its much higher rate of formation (~20,000 s(-1)) compared with its rate of decay (~1,900 s(-1)). Compound I is next converted to a short lived heme d oxoferryl intermediate (compound II) in a phase kinetically matched to the oxidation of heme b(558) before completion of the reaction. The results indicate that cytochrome bd oxidases like the heme-copper oxidases break the O-O bond in a single four-electron transfer without a peroxide intermediate. However, in cytochrome bd oxidases, the fourth electron is donated by the porphyrin moiety rather than by a nearby amino acid. The production of reactive oxygen species by the cytochrome bd oxidase was below the detection level of 1 per 1000 turnovers. We propose that the two classes of terminal oxidases have mechanistically converged to enzymes in which the O-O bond is broken in a single four-electron transfer reaction to safeguard the cell from the formation of reactive oxygen species.
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http://dx.doi.org/10.1074/jbc.M111.333542DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3308821PMC
March 2012

Spectroscopic characterization of cytochrome P450 Compound I.

Arch Biochem Biophys 2011 Mar 30;507(1):44-55. Epub 2010 Dec 30.

Max-Delbrück-Center for Molecular Medicine, Robert-Rössle Strasse 10, Berlin, Germany.

The cytochrome P450 protein-bound porphyrin complex with the iron-coordinated active oxygen atom as Fe(IV)O is called Compound I (Cpd I). Cpd I is the intermediate species proposed to hydroxylate directly the inert carbon-hydrogen bonds of P450 substrates. In the natural reaction cycle of cytochrome P450 Cpd I has not yet been detected, presumably because it is very short-lived. A great variety of experimental approaches has been applied to produce Cpd I artificially aiming to characterize its electronic structure with spectroscopic techniques. In spite of these attempts, none of the spectroscopic studies of the last decades proved capable of univocally identifying the electronic state of P450 Cpd I. Very recently, however, Rittle and Green [9] have shown that Cpd I of CYP119, the thermophilic P450 from Sulfolobus acidocaldarius, is univocally a Fe(IV)O-porphyrin radical with the ferryl iron spin (S=1) antiferromagnetically coupled to the porphyrin radical spin (S'=1/2) yielding a S(tot)=1/2 ground state very similar to Cpd I of chloroperoxidase from Caldariomyces fumago. In this mini-review the efforts to characterize Cpd I of cytochrome P450 by spectroscopic methods are summarized.
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http://dx.doi.org/10.1016/j.abb.2010.12.029DOI Listing
March 2011

Physiological role of the respiratory quinol oxidase in the anaerobic nitrite-reducing methanotroph 'Candidatus Methylomirabilis oxyfera'.

Microbiology (Reading) 2011 Mar 11;157(Pt 3):890-898. Epub 2010 Nov 11.

Centre for Biotechnology, University of Bielefeld, Postfach 10 01 31, D-33501 Bielefeld, Germany.

The anaerobic nitrite-reducing methanotroph 'Candidatus Methylomirabilis oxyfera' ('Ca. M. oxyfera') produces oxygen from nitrite by a novel pathway. The major part of the O(2) is used for methane activation and oxidation, which proceeds by the route well known for aerobic methanotrophs. Residual oxygen may serve other purposes, such as respiration. We have found that the genome of 'Ca. M. oxyfera' harbours four sets of genes encoding terminal respiratory oxidases: two cytochrome c oxidases, a third putative bo-type ubiquinol oxidase, and a cyanide-insensitive alternative oxidase. Illumina sequencing of reverse-transcribed total community RNA and quantitative real-time RT-PCR showed that all four sets of genes were transcribed, albeit at low levels. Oxygen-uptake and inhibition experiments, UV-visible absorption spectral characteristics and EPR spectroscopy of solubilized membranes showed that only one of the four oxidases is functionally produced by 'Ca. M. oxyfera', notably the membrane-bound bo-type terminal oxidase. These findings open a new role for terminal respiratory oxidases in anaerobic systems, and are an additional indication of the flexibility of terminal oxidases, of which the distribution among anaerobic micro-organisms may be largely underestimated.
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http://dx.doi.org/10.1099/mic.0.045187-0DOI Listing
March 2011

Adaptation to a high-tungsten environment: Pyrobaculum aerophilum contains an active tungsten nitrate reductase.

Biochemistry 2010 Nov 21;49(45):9911-21. Epub 2010 Oct 21.

Laboratory of Biotechnology, Delft University of Technology, Julianalaan 67, 2628 BC Delft, The Netherlands.

Nitrate reductases (Nars) belong to the DMSO reductase family of molybdoenzymes. The hyperthermophilic denitrifying archaeon Pyrobaculum aerophilum exhibits nitrate reductase (Nar) activity even at WO(4)(2-) concentrations that are inhibitory to bacterial Nars. In this report, we establish that the enzyme purified from cells grown with 4.5 μM WO(4)(2-) contains W as the metal cofactor but is otherwise identical to the Mo-Nar previously purified from P. aerophilum grown at low WO(4)(2-) concentrations. W is coordinated by a bis-molybdopterin guanine dinucleotide cofactor. The W-Nar has a 2-fold lower turnover number (633 s(-1)) but the same K(m) value for nitrate (56 μM) as the Mo-Nar. Quinol reduction and nitrate oxidation experiments monitored by EPR with both pure W-Nar and mixed W- and Mo-Nar preparations suggest a monodentate ligation by the conserved Asp241 for W(V), while Asp241 acts as a bidentate ligand for Mo(V). Redox titrations of the Mo-Nar revealed a midpoint potential of 88 mV for Mo(V/IV). The E(m) for W(V/IV) of the purified W-Nar was estimated to be -8 mV. This relatively small difference in midpoint potential is consistent with comparable enzyme activities of W- and Mo-Nars. Unlike bacterial Nars, the P. aerophilum Nar contains a unique membrane anchor, NarM, with a single heme of the o(P) type (E(m) = 126 mV). In contrast to bacterial Nars, the P. aerophilum Nar faces the cell's exterior and, hence, does not contribute to the proton motive force. Formate is used as a physiological electron donor. This is the first description of an active W-containing Nar demonstrating the unique ability of hyperthermophiles to adapt to their high-WO(4)(2-) environment.
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http://dx.doi.org/10.1021/bi100974vDOI Listing
November 2010

The millisecond intermediate in the reaction of nitric oxide with oxymyoglobin is an iron(III)--nitrato complex, not a peroxynitrite.

J Am Chem Soc 2009 Jun;131(21):7234-5

Department of Science and Engineering, School of Medicine, Oregon Health & Science University, Beaverton, Oregon 97006-8921, USA.

The dioxygenation of nitric oxide by oxyheme in globin proteins is a major route for NO detoxification in aerobic biological systems. In myoglobin, this reaction is thought to proceed through an iron(III)-bound peroxynitrite before homolytic cleavage of the O-O bond to form an iron(IV)-oxo and NO(2) radical followed by recombination and nitrate production. Single turnover experiments at alkaline pH have revealed the presence of a millisecond high-spin heme intermediate. It is widely presumed that this species is an iron(III)-peroxynitrite species, but detailed characterization of the intermediate is lacking. Using resonance Raman spectroscopy and rapid-freeze quench techniques, we identify the millisecond intermediate as an iron(III)-nitrato complex with a symmetric NO(2) stretch at 1282 cm(-1). Greater time resolution techniques will be required to detect the putative iron(III) peroxynitrite complex.
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http://dx.doi.org/10.1021/ja9026924DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2734454PMC
June 2009

A view on phosphate ester photochemistry by time-resolved solid state NMR. Intramolecular redox reaction of caged ATP.

Phys Chem Chem Phys 2008 Dec 8;10(45):6820-8. Epub 2008 Oct 8.

Biophysical Organic Chemistry/Solid State NMR group, Leiden Institute of Chemistry, Faculty of Mathematics and Natural Sciences, Leiden University, Einsteinweg 55, 2333 CC, Leiden, The Netherlands.

The light-driven intramolecular redox reaction of adenosine-5'-triphosphate-[P3-(1-(2-nitrophenyl)-ethyl)]ester (caged ATP) has been studied in frozen aqueous solution using time-resolved solid state NMR spectroscopy under continuous illumination conditions. Cleavage of the phosphate ester bond leads to 0.3, 1.36, and 6.06 ppm downfield shifts of the alpha-, beta-, and gamma-phosphorus resonances of caged ATP, respectively. The observed rate of ATP formation is 2.4 +/- 0.2 h(-1) at 245 K. The proton released in the reaction binds to the triphosphate moiety of the nascent ATP, causing the upfield shifts of the 31P resonances. Analyses of the reaction kinetics indicate that bond cleavage and proton release are two sequential processes in the solid state, suggesting that the 1-hydroxy,1-(2-nitrosophenyl)-ethyl carbocation intermediate is involved in the reaction. The beta-phosphate oxygen atom of ATP is protonated first, indicating its proximity to the reaction center, possibly within hydrogen bonding distance. The residual linewidth kinetics are interpreted in terms of chemical exchange processes, hydrogen bonding of the beta-phosphate oxygen atom and evolution of the hydrolytic equilibrium at the triphosphate moiety of the nascent ATP. Photoreaction of caged ATP in situ gives an opportunity to study structural kinetics and catalysis of ATP-dependent enzymes by NMR spectroscopy in rotating solids.
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http://dx.doi.org/10.1039/b806677aDOI Listing
December 2008

Structural and biochemical characterization of flavoredoxin from the archaeon Methanosarcina acetivorans.

Biochemistry 2008 Nov 9;47(44):11528-35. Epub 2008 Oct 9.

Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania 16802, USA.

Flavoredoxin is a FMN-containing electron transfer protein that functions in the energy-yielding metabolism of Desulfovibrio gigas of the Bacteria domain. Although characterization of this flavoredoxin is the only one reported, a database search revealed homologues widely distributed in both the Bacteria and Archaea domains that define a novel family. To improve our understanding of this family, a flavoredoxin from Methanosarcina acetivorans of the Archaea domain was produced in Escherichia coli and biochemically characterized, and a high-resolution crystal structure was determined. The protein was shown to be a homodimer with a subunit molecular mass of 21 kDa containing one noncovalently bound FMN per monomer. Redox titration showed an E(m) of -271 mV with two electrons, consistent with no semiquinone observed in the potential range studied, a result suggesting the flavoredoxin functions as a two-electron carrier. However, neither of the obligate two-electron carriers, NAD(P)H and coenzyme F420H2, was a competent electron donor, whereas 2[4Fe-4S] ferredoxin reduced the flavoredoxin. The X-ray crystal structure determined at 2.05 A resolution revealed a homodimer containing one FMN per monomer, consistent with the biochemical characterization. The isoalloxazine ring of FMN was shown buried within a narrow groove approximately 10 A from the positively charged protein surface that possibly facilitates interaction with the negatively charged ferredoxin. The structure provides a basis for predicting the mechanism by which electrons are transferred between ferredoxin and FMN. The FMN is bound with hydrogen bonds to the isoalloxazine ring and electrostatic interactions with the phosphate moiety that, together with sequence analyses of homologues, indicate a novel FMN binding motif for the flavoredoxin family.
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http://dx.doi.org/10.1021/bi801012pDOI Listing
November 2008

Very early reaction intermediates detected by microsecond time scale kinetics of cytochrome cd1-catalyzed reduction of nitrite.

J Biol Chem 2008 Oct 31;283(41):27403-27409. Epub 2008 Jul 31.

Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, United Kingdom. Electronic address:

Paracoccus pantotrophus cytochrome cd(1) is a nitrite reductase found in the periplasm of many denitrifying bacteria. It catalyzes the reduction of nitrite to nitric oxide during the denitrification part of the biological nitrogen cycle. Previous studies of early millisecond intermediates in the nitrite reduction reaction have shown, by comparison with pH 7.0, that at the optimum pH, approximately pH 6, the earliest intermediates were lost in the dead time of the instrument. Access to early time points (approximately 100 micros) through use of an ultra-rapid mixing device has identified a spectroscopically novel intermediate, assigned as the Michaelis complex, formed from reaction of fully reduced enzyme with nitrite. Spectroscopic observation of the subsequent transformation of this species has provided data that demand reappraisal of the general belief that the two subunits of the enzyme function independently.
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http://dx.doi.org/10.1074/jbc.M804493200DOI Listing
October 2008

The associative nature of adenylyl transfer catalyzed by T4 DNA ligase.

Proc Natl Acad Sci U S A 2008 Jun 18;105(25):8563-8. Epub 2008 Jun 18.

Biophysical Organic Chemistry/Solid-State NMR, Leiden Institute of Chemistry, Faculty of Mathematics and Natural Sciences, Leiden University, Einsteinweg 55, 2333CC Leiden, The Netherlands.

DNA ligase seals nicks in dsDNA using chemical energy of the phosphoanhydride bond in ATP or NAD(+) and assistance of a divalent metal cofactor Mg(2+). Molecular details of ligase catalysis are essential for understanding the mechanism of metal-promoted phosphoryl transfer reactions in the living cell responsible for a wide range of processes, e.g., DNA replication and transcription, signaling and differentiation, energy coupling and metabolism. Here we report a single-turnover (31)P solid-state NMR study of adenylyl transfer catalyzed by DNA ligase from bacteriophage T4. Formation of a high-energy covalent ligase-nucleotide complex is triggered in situ by the photo release of caged Mg(2+), and sequentially formed intermediates are monitored by NMR. Analyses of reaction kinetics and chemical-shift changes indicate that the pentacoordinated phosphorane intermediate builds up to 35% of the total reacting species after 4-5 h of reaction. This is direct experimental evidence of the associative nature of adenylyl transfer catalyzed by DNA ligase. NMR spectroscopy in rotating solids is introduced as an analytical tool for recording molecular movies of reaction processes. Presented work pioneers a promising direction in structural studies of biochemical transformations.
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http://dx.doi.org/10.1073/pnas.0709140105DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2438423PMC
June 2008

The role of the conserved tryptophan272 of the Paracoccus denitrificans cytochrome c oxidase in proton pumping.

Authors:
Simon de Vries

Biochim Biophys Acta 2008 Jul-Aug;1777(7-8):925-8. Epub 2008 May 20.

Department of Biotechnology, Delft University of Technology, Julianalaan 67, 2628 BC, Delft, The Netherlands.

The catalytic mechanism of heme-copper oxidases - electron transfer coupled to proton pumping - is not yet fully understood. Single turnover experiments in which fully reduced cytochrome aa(3) from Paracoccus denitrificans reacts with O(2) using the microsecond freeze-hyperquenching sampling technique enabled trapping of transient catalytic intermediates and analysis by low temperature UV-Visible, X-band and Q-band EPR spectroscopy. Our recent findings (Wiertz et al. (2007) J. Biol. Chem. 282, 31580-31591), which show that the strictly conserved W272 is a redox active residue are reviewed here. The W272 forms a tryptophan neutral radical in the transition F-->F(W)-->O(H) in which the novel intermediate F(W) harbors the tryptophan radical. The potential role of W272 in proton pumping is highlighted.
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http://dx.doi.org/10.1016/j.bbabio.2008.05.008DOI Listing
August 2008

Kinetic resolution of a tryptophan-radical intermediate in the reaction cycle of Paracoccus denitrificans cytochrome c oxidase.

J Biol Chem 2007 Oct 30;282(43):31580-91. Epub 2007 Aug 30.

Department of Biotechnology, Delft University of Technology, Julianalaan 67, Delft 2628 BC, The Netherlands.

The catalytic mechanism, electron transfer coupled to proton pumping, of heme-copper oxidases is not yet fully understood. Microsecond freeze-hyperquenching single turnover experiments were carried out with fully reduced cytochrome aa(3) reacting with O(2) between 83 micros and 6 ms. Trapped intermediates were analyzed by low temperature UV-visible, X-band, and Q-band EPR spectroscopy, enabling determination of the oxidation-reduction kinetics of Cu(A), heme a, heme a(3), and of a recently detected tryptophan radical (Wiertz, F. G. M., Richter, O. M. H., Cherepanov, A. V., MacMillan, F., Ludwig, B., and de Vries, S. (2004) FEBS Lett. 575, 127-130). Cu(B) and heme a(3) were EPR silent during all stages of the reaction. Cu(A) and heme a are in electronic equilibrium acting as a redox pair. The reduction potential of Cu(A) is 4.5 mV lower than that of heme a. Both redox groups are oxidized in two phases with apparent half-lives of 57 micros and 1.2 ms together donating a single electron to the binuclear center in each phase. The formation of the heme a(3) oxoferryl species P(R) (maxima at 430 nm and 606 nm) was completed in approximately 130 micros, similar to the first oxidation phase of Cu(A) and heme a. The intermediate F (absorbance maximum at 571 nm) is formed from P(R) and decays to a hitherto undetected intermediate named F(W)(*). F(W)(*) harbors a tryptophan radical, identified by Q-band EPR spectroscopy as the tryptophan neutral radical of the strictly conserved Trp-272 (Trp-272(*)). The Trp-272(*) populates to 4-5% due to its relatively low rate of formation (t((1/2)) = 1.2 ms) and rapid rate of breakdown (t((1/2)) = 60 micros), which represents electron transfer from Cu(A)/heme a to Trp-272(*). The formation of the Trp-272(*) constitutes the major rate-determining step of the catalytic cycle. Our findings show that Trp-272 is a redox-active residue and is in this respect on an equal par to the metallocenters of the cytochrome c oxidase. Trp-272 is the direct reductant either to the heme a(3) oxoferryl species or to Cu (2+)(B). The potential role of Trp-272 in proton pumping is discussed.
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http://dx.doi.org/10.1074/jbc.M705520200DOI Listing
October 2007

Formation of a dinitrosyl iron complex by NorA, a nitric oxide-binding di-iron protein from Ralstonia eutropha H16.

J Biol Chem 2007 Jul 16;282(28):20292-300. Epub 2007 May 16.

Institut für Biologie/Mikrobiologie, Humboldt-Universität zu Berlin, Chausseestrasse 117, 10115 Berlin, Germany.

In Ralstonia eutropha H16, two genes, norA and norB, form a dicistronic operon that is controlled by the NO-responsive transcriptional regulator NorR. NorB has been identified as a membrane-bound NO reductase, but the physiological function of NorA is unknown. We found that, in a NorA deletion mutant, the promoter activity of the norAB operon was increased 3-fold, indicating that NorA attenuates activation of NorR. NorA shows limited sequence similarity to the oxygen carrier hemerythrin, which contains a di-iron center. Indeed, optical and EPR spectroscopy of purified NorA revealed the presence of a di-iron center, which binds oxygen in a similar way as hemerythrin. Diferrous NorA binds two molecules of NO maximally. Unexpectedly, binding of NO to the diferrous NorA required an external reductant. Two different NorA-NO species could be resolved. A minor species (up to 20%) showed an S = (1/2) EPR signal with g( perpendicular) = 2.041, and g( parallel) = 2.018, typical of a paramagnetic dinitrosyl iron complex. The major species was EPR-silent, showing characteristic signals at 420 nm and 750 nm in the optical spectrum. This species is proposed to represent a novel dinitrosyl iron complex of the form Fe(2+)-[NO](2)(2-), i.e. NO is bound as NO(-). The NO binding capacity of NorA in conjunction with its high cytoplasmic concentration (20 mum) suggests that NorA regulates transcription by lowering the free cytoplasmic concentration of NO.
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http://dx.doi.org/10.1074/jbc.M702003200DOI Listing
July 2007
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