Reliable estimation of the kinetic parameters of redox enzymes by taking into account mass transport towards rotating electrodes in protein film voltammetry experiments

International audienceIn Protein Film Voltammetry, a redox enzyme is immobilized on a rotating electrode in a configuration allowing fast, direct electron transfer. This technique is used to probe the mechanism of enzymes by quantitatively interpreting the response in current as a function of the experimental conditions. Limitation by mass transport of the substrate towards the electrode may obscure important features and complicate the analysis of the enzymatic response, so much so that the enzyme has high activity. In this work, we derive equations taking into account mass transport of substrate, for the steady-state current generated by an enzyme following Michaelis-Menten kinetics and immobilized onto a hydrodynamic (e.g. rotating) electrode. We use these equations to model the current response of films of CO-dehydrogenase, a metalloenzyme that catalyzes the oxidation of CO, to transient exposures to its gaseous substrate. We show that neglecting transport yields poor fits and overestimated, unreliable, values of K m (even when using the Koutecky-Levich approximation), whereas taking into account transport yields much better fits and more reliable parameters. We reinterpret previously published data by taking into account transport limitations

Functional and Biochemical Analysis of Chlamydia trachomatis MurC, an Enzyme Displaying UDP-N-Acetylmuramate:Amino Acid Ligase Activity

Chlamydiae are unusual obligate intracellular bacteria that cause serious infections in humans. Chlamydiae contain genes that appear to encode products with peptidoglycan biosynthetic activity. The organisms are also susceptible to antibiotics that inhibit peptidoglycan synthesis. However, chlamydiae do not synthesize detectable peptidoglycan. The paradox created by these observations is known as the chlamydial anomaly. The MurC enzyme of chlamydiae, which is synthesized as a bifunctional MurC-Ddl product, is expected to possess UDP-N-acetylmuramate (UDP-MurNAc):l-alanine ligase activity. In this paper we demonstrate that the MurC domain of the Chlamydia trachomatis bifunctional protein is functionally expressed in Escherichia coli, since it complements a conditional lethal E. coli mutant possessing a temperature-sensitive lesion in MurC. The recombinant MurC domain was overexpressed in and purified from E. coli. It displayed in vitro ATP-dependent UDP-MurNAc:l-alanine ligase activity, with a pH optimum of 8.0 and dependence upon magnesium ions (optimum concentration, 20 mM). Its substrate specificity was studied with three amino acids (l-alanine, l-serine, and glycine); comparable V(max)/K(m) values were obtained. Our results are consistent with the synthesis of a muramic acid-containing polymer in chlamydiae with UDP-MurNAc-pentapeptide as a precursor molecule. However, due to the lack of specificity of MurC activity in vitro, it is not obvious which amino acid is present in the first position of the pentapeptide

Modulation of the RNA polymerase activity by AtcB, a protein associated with a DnaK chaperone network in Shewanella oneidensis

International audienceBacteria possess several molecular pathways to adapt to changing environments and to stress conditions. One of these pathways involves a complex network of chaperone proteins that together control proteostasis. In the aquatic bacterium Shewanella oneidensis, we have recently identified a previously unknown co-chaperone of the DnaK/Hsp70 chaperone system, AtcJ, that is essential for adaptation to low temperatures. AtcJ is encoded in the atcJABC operon, whose products, together with DnaK, form a protein network allowing growth at low temperature. However, how these proteins allow cold adaptation is unknown. Here, we found that AtcB directly interacts with the RNA polymerase and decreases its activity. In addition, AtcB overproduction prevents bacterial growth due to RNA polymerase inhibition. Together, these results suggest that the Atc proteins could direct the DnaK chaperone to the RNA polymerase to sustain life at low temperatures

The mechanism of inhibition by H-2 of H-2-evolution by hydrogenases

By analysing the results of experiments carried out with two FeFe hydrogenases and several "channel mutants'' of a NiFe hydrogenase, we demonstrate that whether or not hydrogen evolution is significantly inhibited by H-2 is not a consequence of active site chemistry, but rather relates to H-2 transport within the enzyme

Steady-State Catalytic Wave-Shapes for 2-Electron Reversible Electrocatalysts and Enzymes

Using direct electrochemistry to learn about the mechanism of electrocatalysts and redox enzymes requires that kinetic models be developed. Here we thoroughly discuss the interpretation of electrochemical signals obtained with adsorbed enzymes and molecular catalysts that can reversibly convert their substrate and product. We derive analytical relations between electrochemical observables (overpotentials for catalysis in each direction, positions, and magnitudes of the features of the catalytic wave) and the characteristics of the catalytic cycle (redox properties of the catalytic intermediates, kinetics of intramolecular and interfacial electron transfer, etc.). We discuss whether or not the position of the wave is determined by the redox potential of a redox relay when intramolecular electron transfer is slow. We demonstrate that there is no simple relation between the reduction potential of the active site and the catalytic bias of the enzyme, defined as the ratio of the oxidative and reductive limiting currents; this explains the recent experimental observation that the catalytic bias of NiFe hydrogenase depends on steps of the catalytic cycle that occur far from the active site [Abou Hamdan et al., J. Am. Chem. Soc. 2012, 134, 8368]. On the experimental side, we examine which models can best describe original data obtained with various NiFe and FeFe hydrogenases, and we illustrate how the presence of an intramolecular electron transfer chain affects the voltammetry by comparing the data obtained with the FeFe hydrogenases from Chlamydomonas reinhardtii and Clostridium acetobutylicum, only one of which has a chain of redox relays. The considerations herein will help the interpretation of electrochemical data previously obtained with various other bidirectional oxidoreductases, and, possibly, synthetic inorganic catalysts