Carole Baffert

Relating diffusion along the substrate tunnel and oxygen sensitivity in hydrogenase

In hydrogenases and many other redox enzymes, the buried active site is connected to the solvent by a molecular channel whose structure may determine the enzymes selectivity with respect to substrate and inhibitors. The role of these channels has been addressed using crystallography and molecular dynamics, but kinetic data are scarce. Using protein film voltammetry, we determined and then compared the rates of inhibition by CO and O2 in ten NiFe hydrogenase mutants and two FeFe hydrogenases. We found that the rate of inhibition by CO is a good proxy of the rate of diffusion of O2 toward the active site. Modifying amino acids whose side chains point inside the tunnel can slow this rate by orders of magnitude. We quantitatively define the relations between diffusion, the Michaelis constant for H2 and rates of inhibition, and we demonstrate that certain enzymes are slowly inactivated by O2 because access to the active site is slow.

SOAS: a free program to analyze electrochemical data and other one-dimensional signals

This paper describes an open source program called SOAS, which we developed with the aim of analysing one-dimensional signals. It offers a large set of commands for handling voltammetric and chronoamperometric data, including smoothing signals, differentiation, subtracting baselines, fitting current responses, measuring limiting currents, and searching for peak positions. Although emphasis is on the analysis of electrochemical signals, particularly protein film voltammetry data, SOAS may also prove useful for processing spectra. This free program is available by download from the Internet, and can be installed on computers running any flavor of Unix or Linux, most easily on MacOS X.

The oxidative inactivation of FeFe hydrogenase reveals the flexibility of the H-cluster

Nature is a valuable source of inspiration in the design of catalysts, and various approaches are used to elucidate the mechanism of hydrogenases, the enzymes that oxidize or produce H2. In FeFe hydrogenases, H2 oxidation occurs at the H-cluster, and catalysis involves H2 binding on the vacant coordination site of an iron centre. Here, we show that the reversible oxidative inactivation of this enzyme results from the binding of H2 to coordination positions that are normally blocked by intrinsic CO ligands. This flexibility of the coordination sphere around the reactive iron centre confers on the enzyme the ability to avoid harmful reactions under oxidizing conditions, including exposure to O2. The versatile chemistry of the diiron cluster in the natural system might inspire the design of novel synthetic catalysts for H2 oxidation.

O2-independent formation of the inactive states of NiFe hydrogenase

We studied the mechanism of aerobic inactivation of Desulfovibrio fructosovorans nickel-iron (NiFe) hydrogenase by quantitatively examining the results of electrochemistry, EPR and FTIR experiments. They suggest that, contrary to the commonly accepted mechanism, the attacking O(2) is not incorporated as an active site ligand but, rather, acts as an electron acceptor. Our findings offer new ways toward the understanding of O(2) inactivation and O(2) tolerance in NiFe hydrogenases.

Covalent attachment of FeFe hydrogenases to carbon electrodes for direct electron transfer.

Direct electron transfer between enzymes and electrodes is now commonly achieved, but obtaining protein films that are very stable may be challenging. This is particularly crucial in the case of hydrogenases, the enzymes that catalyze the biological conversion between dihydrogen and protons, because the instability of the hydrogenase films may prevent the use of these enzymes as electrocatalysts of H(2) oxidation and production in biofuel cells and photoelectrochemical cells. Here we show that two different FeFe hydrogenases (from Chamydomonas reinhardtii and Clostridium acetobutylicum) can be covalently attached to functionalized pyrolytic graphite electrodes using peptidic coupling. In both cases, a surface patch of lysine residues makes it possible to favor an orientation that is efficient for fast, direct electron transfer. High hydrogen-oxidation current densities are maintained for up to one week, the only limitation being the intrinsic stability of the enzyme. We also show that covalent attachment has no effect on the catalytic properties of the enzyme, which means that this strategy can also used be for electrochemical studies of the catalytic mechanism.

The quest for a functional substrate access tunnel in FeFe hydrogenase

We investigated di-hydrogen transport between the solvent and the active site of FeFe hydrogenases. Substrate channels supposedly exist and serve various functions in certain redox enzymes which use or produce O2, H2, NO, CO, or N2, but the preferred paths have not always been unambiguously identified, and whether a continuous, permanent channel is an absolute requirement for transporting diatomic molecules is unknown. Here, we review the literature on gas channels in proteins and enzymes and we report on the use of site-directed mutagenesis and various kinetic methods, which proved useful for characterizing substrate access to the active site of NiFe hydrogenase to test the putative static H2 channel of FeFe hydrogenases. We designed 8 mutations in attempts to interfere with intramolecular diffusion by remodeling this putative route in Clostridium acetobutylicum FeFe hydrogenase, and we observed that none of them has a strong effect on any of the enzymes kinetic properties. We suggest that H2 may diffuse either via transient cavities, or along a conserved water-filled channel. Nitrogenase sets a precedent for the involvement of a hydrophilic channel to conduct hydrophobic molecules.

CO Disrupts the Reduced H-Cluster of FeFe Hydrogenase. A Combined DFT and Protein Film Voltammetry Study

Carbon monoxide is often described as a competitive inhibitor of FeFe hydrogenases, and it is used for probing H(2) binding to synthetic or in silico models of the active site H-cluster. Yet it does not always behave as a simple inhibitor. Using an original approach which combines accurate electrochemical measurements and theoretical calculations, we elucidate the mechanism by which, under certain conditions, CO binding can cause permanent damage to the H-cluster. Like in the case of oxygen inhibition, the reaction with CO engages the entire H-cluster, rather than only the Fe(2) subsite.

Carboxylate ligands drastically enhance the rates of oxo exchange and hydrogen peroxide disproportionation by oxo manganese compounds of potential biological significance.

To mimic the carboxylate-rich active site of the manganese catalases more closely we introduced carboxylate groups into dimanganese complexes in place of nitrogen ligands. The series of dimanganese(III,IV) complexes of tripodal ligands [Mn(2)(L)(2)(O)(2)](3+/+/-/3-) was extended from those of tpa (1) and H(bpg) (2) to those of H(2)(pda) (3) and H(3)(nta) (4) (tpa=tris-picolylamine, H(bpg)=bis-picolylglycylamine, H(2)(pda)=picolyldiglycylamine, H(3)(nta)=nitrilotriacetic acid). While 3 [Mn(2)(pda)(2)(O)(2)][Na(H(2)O)(3)] could be synthesized at -20 degrees C and characterized in the solid state, 4 [Mn(2)(nta)(2)(O)(2)](3-) could be obtained and studied only in solution at -60 degrees C. A new synthetic procedure for the dimanganese(III,III) complexes was devised, using stoichiometric reduction of the dimanganese(III,IV) precursor by the benzil radical with EPR monitoring. This enabled the preparation of the parent dimanganese(III,III) complex 5 [Mn(2)(tpa)(2)(O)(2)](ClO(4))(2), which was structurally characterized. The UV/visible, IR, EPR, magnetic, and electrochemical properties of complexes 1-3 and 5 were analyzed to assess the electronic changes brought about by the carboxylate replacement of pyridine ligands. The kinetics of the oxo ligand exchanges with labeled water was examined in acetonitrile solution. A dramatic effect of the number of carboxylates was evidenced. Interestingly, the influence of the second carboxylate substitution differs from that of the first one probably because this substitution occurs on an out-of-plane coordination while the former occurs in the plane of the [Mn(2)O(2)] core. Indeed, on going from 1 to 3 the exchange rate was increased by a factor of 50. Addition of triethylamine caused a rate increase for 1, but not for 3. The abilities of 1-3 to disproportionate H(2)O(2) were assessed volumetrically. The disproportionation exhibited a sensitivity corresponding to the carboxylate substitution. These observations strongly suggest that the carboxylate ligands in 2 and 3 act as internal bases.

Trinuclear terpyridine frustrated spin system with a Mn(IV)3O4 core: synthesis, physical characterization, and quantum chemical modeling of its magnetic properties.

The trinuclear oxo bridged manganese cluster, [Mn(IV)(3)O(4)(terpy)(terpyO(2))(2)(H(2)O)](S(2)O(8))(2) (5) (terpy = 2,2:2,6-terpyridine and terpyO(2) = 2,2:2,6-terpyridine 1,1-dioxide), was isolated in an acidic aqueous medium from the reaction of MnSO(4), terpy, and oxone as chemical oxidant. The terpyO(2) ligands were generated in situ during the synthesis by partial oxidation of terpy. The complex crystallizes in the monoclinic space group P21/n with a = 14.251(5) A, b = 15.245(5) A, c = 24.672(5) A, alpha = 90.000(5) degrees, beta = 92.045(5) degrees, gamma = 90.000(5) degrees, and Z = 4. The triangular {Mn(IV)(3)O(4)}(4+) core observed in this complex is built up of a basal Mn(mu-O)(2)Mn unit where each Mn ion is linked to an apical Mn ion via mono(mu-O) bridges. The facial coordination of the two tridentate terpyO(2) ligands to the Mn(mu-O)(2)Mn unit allows the formation of the triangular core. 5 is also the first structurally characterized Mn complex with polypyridinyl N-oxide ligands. The variable-temperature magnetic susceptibility data for this complex, in the range of 10-300 K, are consistent with an S = 1/2 ground state and were fit using the spin Hamiltonian H(eff) with S(1) = S(2) = S(3) = 3/2, J(a) = -37 (+/-0.5) and J(b) = -53 (+/-1) cm(-1), where J(a) and J(b) are exchange constants through the mono-mu-oxo and the di-mu-oxo bridges, respectively. The doublet ground spin state of 5 is confirmed by EPR spectroscopic measurements. Density functional theory (DFT) calculations based on the broken symmetry approach reproduce the magnetic properties of 5 very well (calculated values: J(a) = -39.4 and J(b) = -55.9 cm(-1)), thus confirming the capability of this quantum chemical method for predicting the magnetic behavior of clusters involving more than two metal ions. The nature of the ground spin state of the magnetic {Mn(IV)(3)O(4)}(4+) core and the role of ancillary ligands on the magnitude of J are also discussed.

Correcting for Electrocatalyst Desorption and Inactivation in Chronoamperometry Experiments

Chronoamperometric experiments with adsorbed electrocatalysts are commonly performed either for analytical purposes or for studying the catalytic mechanism of a redox enzyme. In the context of amperometric sensors, the current may be recorded as a function of time while the analyte concentration is being increased to determine a linearity range. In mechanistic studies of redox enzymes, chronoamperometry proved powerful for untangling the effects of electrode potential and time, which are convoluted in cyclic voltammetric measurements, and for studying the energetics and kinetics of inhibition. In all such experiments, the fact that the catalysts coverage and/or activity decreases over time distorts the data. This may hide meaningful features, introduce systematic errors, and limit the accuracy of the measurements. We propose a general and surprisingly simple method for correcting for electrocatalyst desorption and inactivation, which greatly increases the precision of chronoamperometric experiments. Rather than subtracting a baseline, this consists in dividing the current, either by a synthetic signal that is proportional to the instant electroactive coverage or by the signal recorded in a control experiment. In the latter, the change in current may result from film loss only or from film loss plus catalyst inactivation. We describe the different strategies for obtaining the control signal by analyzing various data recorded with adsorbed redox enzymes: nitrate reductase, NiFe hydrogenase, and FeFe hydrogenase. In each case we discuss the trustfulness and the benefit of the correction. This method also applies to experiments where electron transfer is mediated, rather than direct, providing the current is proportional to the time-dependent concentration of catalyst.