Dynamics Properties of Photosynthetic Microorganisms Probed by Incoherent Neutron Scattering.

Studies on the dynamical properties of photosynthetic membranes of land plants and purple bacteria have been previously performed by neutron spectroscopy, revealing a tight coupling between specific photochemical reactions and macromolecular dynamics. Here, we probed the intrinsic dynamics of biotechnologically useful mutants of the green alga Chlamydomonas reinhardtii by incoherent neutron scattering coupled with prompt chlorophyll fluorescence experiments. We brought to light that single amino acid replacements in the plastoquinone (PQ)-binding niche of the photosystem II D1 protein impair electron transport (ET) efficiency between quinones and confer increased flexibility to the host membranes, expanding to the entire cells. Hence, a more flexible environment in the PQ-binding niche has been associated to a less efficient ET. A similar function/dynamics relationship was also demonstrated in Rhodobacter sphaeroides reaction centers having inhibited ET, indicating that flexibility at the quinones region plays a crucial role in evolutionarily distant organisms. Instead, a different functional/dynamical correlation was observed in algal mutants hosting a single amino acid replacement residing in a D1 domain far from the PQ-binding niche. Noteworthy, this mutant displayed the highest degree of flexibility, and besides having a nativelike ET efficiency in physiological conditions, it acquired novel, to our knowledge, phenotypic traits enabling it to preserve a high maximal quantum yield of photosystem II photochemistry in extreme habitats. Overall, in the nanosecond timescale, the degree of the observed flexibility is related to the mutation site; in the picosecond timescale, we highlighted the presence of a more pronounced dynamic heterogeneity in all mutants compared to the native cells, which could be related to a marked chemically heterogeneous environment.

Utility of fluorescent heme analogue ZnPPIX to monitor conformational heterogeneity in vertebrate hexa-coordinated globins.

Here, we report the preparation and photo-physical characterization of hexa-coordinated vertebrate globins, human neuroglobin (hNgb) and cytoglobin (hCygb), with the native iron protoporphyrin IX (FePPIX) cofactor replaced by a fluorescent isostructural analogue, zinc protoporphyrin IX (ZnPPIX). To facilitate insertion of ZnPPIX into hexa-coordinated globins, apoproteins prepared via butanone extraction were unfolded by the addition of GuHCl and subsequently slowly refolded in the presence of ZnPPIX. The absorption/emission spectra of ZnPPIX reconstituted hCygb are similar to those observed for ZnPPIX reconstituted myoglobin whereas the absorption and emission spectra of ZnPPIX reconstituted hNgb are blue shifted by ∼2 nm. Different steady state absorption and emission properties of ZnPPIX incorporated in hCygb and hNgb are consistent with distinct hydrogen bonding interactions between ZnPPIX and the globin matrix. The fluorescence lifetime of ZnPPIX in hexa-coordinated globins is bimodal pointing towards increased heterogeneity of the heme binding cavity in hCygb and hNgb. ZnPPIX reconstituted Ngb binds to cytochrome c with the same affinity as reported for the native protein, suggesting that fluorescent analogues of Cygb and Ngb can be readily employed to monitor interactions between vertebrate hexa-coordinated globins and other proteins.

Role of Ionic Strength and pH in Modulating Thermodynamic Profiles Associated with CO Escape from Rice Nonsymbiotic Hemoglobin 1.

Type 1 nonsymbiotic hemoglobins are found in a wide variety of land plants and exhibit very high affinities for exogenous gaseous ligands. These proteins are presumed to have a role in protecting plant cells from oxidative stress under etiolated/hypoxic conditions through NO dioxygenase activity. In this study we have employed photoacoustic calorimetry, time-resolved absorption spectroscopy, and classical molecular dynamics simulations in order to elucidate thermodynamics, kinetics, and ligand migration pathways upon CO photodissociation from WT and a H73L mutant of type 1 nonsymbiotic hemoglobin from Oryza sativa (rice). We observe a temperature dependence of the resolved thermodynamic parameters for CO photodissociation from CO-rHb1 which we attribute to temperature dependent formation of a network of electrostatic interactions in the vicinity of the heme propionate groups. We also observe slower ligand escape from the protein matrix under mildly acidic conditions in both the WT and H73L mutant (τ = 134 ± 19 and 90 ± 15 ns). Visualization of transient hydrophobic channels within our classical molecular dynamics trajectories allows us to attribute this phenomenon to a change in the ligand migration pathway which occurs upon protonation of the distal His73, His117, and His152. Protonation of these residues may be relevant to the functioning of the protein in vivo given that etiolation/hypoxia can cause a decrease in intracellular pH in plant cells.

Stigmatellin probes the electrostatic potential in the QB site of the photosynthetic reaction center.

The electrostatic potential in the secondary quinone (QB) binding site of the reaction center (RC) of the photosynthetic bacterium Rhodobacter sphaeroides determines the rate and free energy change (driving force) of electron transfer to QB. It is controlled by the ionization states of residues in a strongly interacting cluster around the QB site. Reduction of the QB induces change of the ionization states of residues and binding of protons from the bulk. Stigmatellin, an inhibitor of the mitochondrial and photosynthetic respiratory chain, has been proven to be a unique voltage probe of the QB binding pocket. It binds to the QB site with high affinity, and the pK value of its phenolic group monitors the local electrostatic potential with high sensitivity. Investigations with different types of detergent as a model system of isolated RC revealed that the pK of stigmatellin was controlled overwhelmingly by electrostatic and slightly by hydrophobic interactions. Measurements showed a high pK value (>11) of stigmatellin in the QB pocket of the dark-state wild-type RC, indicating substantial negative potential. When the local electrostatics of the QB site was modulated by a single mutation, L213Asp → Ala, or double mutations, L213Asp-L212Glu → Ala-Ala (AA), the pK of stigmatellin dropped to 7.5 and 7.4, respectively, which corresponds to a >210 mV increase in the electrostatic potential relative to the wild-type RC. This significant pK drop (ΔpK > 3.5) decreased dramatically to (ΔpK > 0.75) in the RC of the compensatory mutant (AA+M44Asn → AA+M44Asp). Our results indicate that the L213Asp is the most important actor in the control of the electrostatic potential in the QB site of the dark-state wild-type RC, in good accordance with conclusions of former studies using theoretical calculations or light-induced charge recombination assay.

Reduction of the internal disulfide bond between Cys 38 and 83 switches the ligand migration pathway in cytoglobin.

Despite the similar tertiary structure between cytoglobin (Cygb) and myoglobin, several structural features indicate a distinct mechanism of Cygb interactions with exogenous ligands. Here we present a spectroscopic investigation of the dynamics and thermodynamics of structural changes associated with the exogenous ligand migration between the solvent and the heme active site in Cygb with reduced and oxidized Cys 38 and Cys 83 side-chains (Cygb(ox) and Cygb(red), respectively). Photo-acoustic and transient absorption data show that disulfide bond formation alters the ligand migration pathway(s) as evident from the distinct geminate quantum yields (Φgem=0.35 for Cygb(ox) and Φgem=0.63 for Cygb(red)) and rate constants for bimolecular CO rebinding. Moreover, ligand escape from the protein matrix is fast (<40ns) and coupled with an enthalpy change of 18±2kcalmol(-1) in Cygb(red), whereas the disulfide bridge formation promotes a biphasic ligand escape associated with an overall enthalpy change of 9±4kcalmol(-1). These results demonstrate that the disulfide bond connecting helix E and helix B modulates the conformational dynamics in Cygb including the size and energy barrier between the internal hydrophobic sites. Based on the comparison of the thermodynamic profiles for CO photo-dissociation from Cygb, myoglobin, and neuroglobin we propose that in Cygb(red) the photo-dissociated ligand escapes through the hydrophobic tunnel, whereas the CO preferably migrates through the His64 gate in Cygb(ox) suggesting that Cygb's physiological role may vary in response to intracellular redox conditions.

Heme orientation modulates histidine dissociation and ligand binding kinetics in the hexacoordinated human neuroglobin.

Neuroglobin (Ngb) is a globin present in the brain and retina of mammals. This hexacoordinated hemoprotein binds small diatomic molecules, albeit with lower affinity compared with other globins. Another distinctive feature of most mammalian Ngb is their ability to form an internal disulfide bridge that increases ligand affinity. As often seen for prosthetic heme b containing proteins, human Ngb exhibits heme heterogeneity with two alternative heme orientations within the heme pocket. To date, no details are available on the impact of heme orientation on the binding properties of human Ngb and its interplay with the cysteine oxidation state. In this work, we used (1)H NMR spectroscopy to probe the cyanide binding properties of different Ngb species in solution, including wild-type Ngb and the single (C120S) and triple (C46G/C55S/C120S) mutants. We demonstrate that in the disulfide-containing wild-type protein cyanide ligation is fivefold faster for one of the two heme orientations (the A isomer) compared with the other isomer, which is attributed to the lower stability of the distal His64-iron bond and reduced steric hindrance at the bottom of the cavity for heme sliding in the A conformer. We also attribute the slower cyanide reactivity in the absence of a disulfide bridge to the tighter histidine-iron bond. More generally, enhanced internal mobility in the CD loop bearing the disulfide bridge hinders access of the ligand to heme iron by stabilizing the histidine-iron bond. The functional impact of heme disorder and cysteine oxidation state on the properties of the Ngb ligand is discussed.

Conformational dynamics in human neuroglobin: effect of His64, Val68, and Cys120 on ligand migration.

Neuroglobin belongs to the family of hexacoordinate hemoglobins and has been implicated in the protection of neuronal tissue under hypoxic and ischemic conditions. Here we present transient absorption and photoacoustic calorimetry studies of CO photodissociation and bimolecular rebinding to neuroglobin focusing on the ligand migration process and the role of distal pocket residues (His64 and Val68) and two Cys residues (Cys55 and Cys120). Our results indicate that His64 has a minor impact on the migration of CO between the distal heme pocket and protein exterior, whereas the Val68 side chain regulates the transition of the photodissociated ligand between the distal pocket and internal hydrophobic cavities, which is evident from the increased geminate quantum yield in this mutated protein (Φ(gem) = 0.32 for WT and His64Gln, and Φ(gem) = 0.85 for Val68Phe). The interface between helix G and the A-B loop provides an escape pathway for the photodissociated ligand, which is evident from a decrease in the reaction enthalpy for the transition between the CO-bound hNgb and five-coordinate hNgb in the Cys120Ser mutant (ΔH = -3 ± 4 kcal mol(-1)) compared to that of the WT protein (ΔH = 20 ± 4 kcal mol(-1)). The extensive electrostatic/hydrogen binding network that includes heme propionate groups, Lys67, His64, and Tyr44 not only restricts the heme binding but also modulates the energetics of binding of CO to the five-coordinate hNgb as substitution of His64 with Gln leads to an endothermic association of CO with the five-coordinate hNgb (ΔH = 6 ± 3 kcal mol(-1)).

Frontier residues lining globin internal cavities present specific mechanical properties.

The internal cavity matrix of globins plays a key role in their biological function. Previous studies have already highlighted the plasticity of this inner network, which can fluctuate with the proteins breathing motion, and the importance of a few key residues for the regulation of ligand diffusion within the protein. In this Article, we combine all-atom molecular dynamics and coarse-grain Brownian dynamics to establish a complete mechanical landscape for six different globins chain (myoglobin, neuroglobin, cytoglobin, truncated hemoglobin, and chains α and ß of hemoglobin). We show that the rigidity profiles of these proteins can fluctuate along time, and how a limited set of residues present specific mechanical properties that are related to their position at the frontier between internal cavities. Eventually, we postulate the existence of conserved positions within the globin fold, which form a mechanical nucleus located at the center of the cavity network, and whose constituent residues are essential for controlling ligand migration in globins.

Probing the role of the internal disulfide bond in regulating conformational dynamics in neuroglobin.

In this report, we demonstrate that the internal disulfide bridge in human neuroglobin modulates structural changes associated with ligand photo-dissociation from the heme active site. This is evident from time-resolved photothermal studies of CO photo-dissociation, which reveal a 13.4+/-0.9 mL mol(-1) volume expansion upon ligand photo-release from human neuroglobin, whereas the CO dissociation from rat neuroglobin leads to a significantly smaller volume change (DeltaV=4.6+/-0.3 mL mol(-1)). Reduction of the internal disulfide bond in human neuroglobin leads to conformational changes (reflected by DeltaV) nearly identical to those observed for rat Ngb. Our data favor the hypothesis that the disulfide bond between Cys46 and Cys55 modulates the functioning of human neuroglobin.

Relating the diffusion of small ligands in human neuroglobin to its structural and mechanical properties.

Neuroglobin (Ngb), a recently discovered member of the globin family, is overexpressed in the brain tissues over oxygen deprivation. Unlike more classical globins, such as myoglobin and hemoglobin, it is characterized by a hexacoordinated heme, and its physiological role is still unknown, despite the numerous investigations made on the protein in recent years. Another important specific feature of human Ngb is the presence of two cysteine residues (Cys46 and Cys55), which are known to form an intramolecular disulfide bridge. Since previous work on human Ngb reported that its ligand binding properties could be controlled by the coordination state of the Fe(2+) atom (in the heme moiety) and the redox state of the thiol groups, we choose to develop a simulation approach combining coarse-grain Brownian dynamics and all-atom molecular dynamics and metadynamics. We have studied the diffusion of small ligands (CO, NO, and O(2)) in the globin internal cavity network for various states of human Ngb. Our results show how the structural and mechanical properties of the protein can be related to the ligand migration pathway, which can be extensively modified when changing the thiol's redox state and the iron's coordination state. We suggest that ligand binding is favored in the pentacoordinated species bearing an internal disulfide bridge.

M234Glu is a component of the proton sponge in the reaction center from photosynthetic bacteria.

Bacterial reaction centers use light energy to couple the uptake of protons to the successive semi-reduction of two quinones, namely Q(A) and Q(B). These molecules are situated symmetrically in regard to a non-heme iron atom. Four histidines and one glutamic acid, M234Glu, constitute the five ligands of this atom. By flash-induced absorption spectroscopy and delayed fluorescence we have studied in the M234EH and M234EL variants the role played by this acidic residue on the energetic balance between the two quinones as well as in proton uptake. Delayed fluorescence from the P(+)Q(A)(-) state (P is the primary electron donor) and temperature dependence of the rate of P(+)Q(A)(-) charge recombination that are in good agreement show that in the two RC variants, both Q(A)(-) and Q(B)(-) are destabilized by about the same free energy amount: respectively approximately 100 +/- 5 meV and 90 +/- 5 meV for the M234EH and M234EL variants, as compared to the WT. Importantly, in the M234EH and M234EL variants we observe a collapse of the high pH band (present in the wild-type reaction center) of the proton uptake amplitudes associated with formation of Q(A)(-) and Q(B)(-). This band has recently been shown to be a signature of a collective behaviour of an extended, multi-entry, proton uptake network. M234Glu seems to play a central role in the proton sponge-like system formed by the RC protein.

Mechanism of Mg2+ binding in the Na+,K+-ATPase.

The Mg(2+) dependence of the kinetics of the phosphorylation and conformational changes of Na(+),K(+)-ATPase was investigated via the stopped-flow technique using the fluorescent label RH421. The enzyme was preequilibrated in buffer containing 130 mM NaCl to stabilize the E1(Na(+))(3) state. On mixing with ATP, a fluorescence increase was observed. Two exponential functions were necessary to fit the data. Both phases displayed an increase in their observed rate constants with increasing Mg(2+) to saturating values of 195 (+/- 6) s(-1) and 54 (+/- 8) s(-1) for the fast and slow phases, respectively. The fast phase was attributed to enzyme conversion into the E2MgP state. The slow phase was attributed to relaxation of the dephosphorylation/rephosphorylation (by ATP) equilibrium and the buildup of some enzyme in the E2Mg state. Taking into account competition from free ATP, the dissociation constant (K(d)) of Mg(2+) interaction with the E1ATP(Na(+))(3) state was estimated as 0.069 (+/- 0.010) mM. This is virtually identical to the estimated value of the K(d) of Mg(2+)-ATP interaction in solution. Within the enzyme-ATP-Mg(2+) complex, the actual K(d) for Mg(2+) binding can be attributed primarily to complexation by ATP itself, with no apparent contribution from coordination by residues of the enzyme environment in the E1 conformation.

The local electric field within phospholipid membranes modulates the charge transfer reactions in reaction centres.

Three different cholesterol derivatives and phloretin, known to affect the local electric field in phospholipid membranes, have been introduced into Rhodobacter sphaeroides reaction centre-containing phospholipid liposomes. We show that cholesterol and 6-ketocholestanol significantly slow down the interquinone first electron transfer (approximately 10 times), whereas phloretin and 5-cholesten-3beta-ol-7-one leave the kinetics essentially unchanged. Interestingly, the two former compounds have been shown to increase the dipole potential, whereas the two latter decrease it. We also measured in isolated RCs the rates of the electron and proton transfers at the first flash. Over the pH range 7-10.5 both reactions display biphasic behaviors with nearly superimposable rates and amplitudes, suggesting that the gating process limiting the first electron transfer is indeed the coupled proton entry. We therefore interpret the effects of cholesterol and 6-ketocholestanol as due to dipole concentration producing an increased free energy barrier for protons to enter the protein perpendicular to the membrane. We also report for the first time in R. sphaeroides RCs, at room temperature, a biphasicity of the P(+)Q(A)(-) charge recombination, induced by the presence of cholesterol derivatives in proteoliposomes. We propose that these molecules decrease the equilibration time between two RC conformations, therefore revealing their presence.

Proton-transfer pathways in photosynthetic reaction centers analyzed by profile hidden markov models and network calculations.

In the bacterial reaction center (bRC) of Rhodobacter sphaeroides, the key residues of proton transfer to the secondary quinone (Q(B)) are known. Also, several possible proton entry points and proton-transfer pathways have been proposed. However, the mechanism of the proton transfer to Q(B) remains unclear. The proton transfer to Q(B) in the bRC of Blastochloris viridis is less explored. To analyze whether the bRCs of different species use the same key residues for proton transfer to Q(B), we determined the conservation of these residues. We performed a multiple-sequence alignment based on profile hidden Markov models. Residues involved in proton transfer but not located at the protein surface are conserved or are only exchanged to functionally similar amino acids, whereas potential proton entry points are not conserved to the same extent. The analysis of the hydrogen-bond network of the bRC from R. sphaeroides and that from B. viridis showed that a large network connects Q(B) with the cytoplasmic region in both bRCs. For both species, all non-surface key residues are part of the network. However, not all proton entry points proposed for the bRC of R. sphaeroides are included in the network in the bRC of B. viridis. From our analysis, we could identify possible proton entry points. These proton entry points differ between the two bRCs. Together, the results of the conservation analysis and the hydrogen-bond network analysis make it likely that the proton transfer to Q(B) is not mediated by distinct pathways but by a large hydrogen-bond network.

Profile hidden Markov models for analyzing similarities and dissimilarities in the bacterial reaction center and photosystem II.

The bacterial photosynthetic reaction center is the evolutionary ancestor of the Photosystem II reaction center. These proteins share the same fold and perform the same biological function. Nevertheless, the details of their molecular reaction mechanism differ. It is of significant biological and biochemical interest to determine which functional characteristics are conserved at the level of the protein sequences. Since the level of sequence identity between the bacterial photosynthetic reaction center and Photosystem II is low, a progressive multiple-sequence alignment leads to errors in identifying the conserved residues. In such a situation, profile hidden Markov models (pHMM) can be used to obtain reliable multiple-sequence alignments. We therefore constructed the pHMM with the help of a sequence alignment based on a structural superposition of both proteins. To validate the multiple-sequence alignments obtained with the pHMM, the conservation of residues with known functional importance was examined. Having confirmed the correctness of the multiple-sequence alignments, we analyzed the conservation of residues involved in hydrogen bonding and redox potential tuning of the cofactors. Our analysis reveals similarities and dissimilarities between the bacterial photosynthetic reaction center and Photosystem II at the protein sequence level, hinting at different charge separation and charge transfer mechanisms. The conservation analysis that we perform in this paper can be considered as a model for analyzing the conservation in proteins with a low level of sequence identity.

ATP binding equilibria of the Na(+),K(+)-ATPase.

Reported values of the dissociation constant, K(d), of ATP with the E1 conformation of the Na(+),K(+)-ATPase fall in two distinct ranges depending on how it is measured. Equilibrium binding studies yield values of 0.1-0.6 microM, whereas presteady-state kinetic studies yield values of 3-14 microM. It is unacceptable that K(d) varies with the experimental method of its determination. Using simulations of the expected equilibrium behavior for different binding models based on thermodynamic data obtained from isothermal titration calorimetry we show that this apparent discrepancy can be explained in part by the presence in presteady-state kinetic studies of excess Mg(2+) ions, which compete with the enzyme for the available ATP. Another important contributing factor is an inaccurate assumption in the majority of presteady-state kinetic studies of a rapid relaxation of the ATP binding reaction on the time scale of the subsequent phosphorylation. However, these two factors alone are insufficient to explain the previously observed presteady-state kinetic behavior. In addition one must assume that there are two E1-ATP binding equilibria. Because crystal structures of P-type ATPases indicate only a single bound ATP per alpha-subunit, the only explanation consistent with both crystal structural and kinetic data is that the enzyme exists as an (alphabeta)(2) diprotomer, with protein-protein interactions between adjacent alpha-subunits producing two ATP affinities. We propose that in equilibrium measurements the measured K(d) is due to binding of ATP to one alpha-subunit, whereas in presteady-state kinetic studies, the measured apparent K(d) is due to the binding of ATP to both alpha-subunits within the diprotomer.

Probing the flexibility of the bacterial reaction center: the wild-type protein is more rigid than two site-specific mutants.

Experimental and theoretical studies have stressed the importance of flexibility for protein function. However, more local studies of protein dynamics, using temperature factors from crystallographic data or elastic models of protein mechanics, suggest that active sites are among the most rigid parts of proteins. We have used quasielastic neutron scattering to study the native reaction center protein from the purple bacterium Rhodobacter sphaeroides, over a temperature range of 4-260 K, in parallel with two nonfunctional mutants both carrying the mutations L212Glu/L213Asp --> Ala/Ala (one mutant carrying, in addition, the M249Ala --> Tyr mutation). The so-called dynamical transition temperature, Td, remains the same for the three proteins around 230 K. Below Td the mean square displacement, u2, and the dynamical structure factor, S(Q,omega), as measured respectively by backscattering and time-of-flight techniques are identical. However, we report that above Td, where anharmonicity and diffusive motions take place, the native protein is more rigid than the two nonfunctional mutants. The higher flexibility of both mutant proteins is demonstrated by either their higher u2 values or the notable quasielastic broadening of S(Q,omega) that reveals the diffusive nature of the motions involved. Remarkably, we demonstrate here that in proteins, point genetic mutations may notably affect the overall protein dynamics, and this effect can be quantified by neutron scattering. Our results suggest a new direction of investigation for further understanding of the relationship between fast dynamics and activity in proteins. Brownian dynamics simulations we have carried out are consistent with the neutron experiments, suggesting that a rigid core within the native protein is specifically softened by distant point mutations. L212Glu, which is systematically conserved in all photosynthetic bacteria, seems to be one of the key residues that exerts a distant control over the rigidity of the core of the protein.

pH modulates the quinone position in the photosynthetic reaction center from Rhodobacter sphaeroides in the neutral and charge separated states.

The structure of the photosynthetic reaction-center from Rhodobacter sphaeroides has been determined at four different pH values (6.5, 8.0, 9.0, 10.0) in the neutral and in charge separated states. At pH 8.0, in the neutral state, we obtain a resolution of 1.87 A, which is the best ever reported for the bacterial reaction center protein. Our crystallographic data confirm the existence of two different binding positions of the secondary quinone (QB). We observe a new orientation of QB in its distal position, which shows no ring-flip compared to the orientation in the proximal position. Datasets collected for the different pH values show a pH-dependence of the population of the proximal position. The new orientation of QB in the distal position and the pH-dependence could be confirmed by continuum electrostatics calculations. Our calculations are in agreement with the experimentally observed proton uptake upon charge separation. The high resolution of our crystallographic data allows us to identify new water molecules and external residues being involved in two previously described hydrogen bond proton channels. These extended proton-transfer pathways, ending at either of the two oxo-groups of QB in its proximal position, provide additional evidence that ring-flipping is not required for complete protonation of QB upon reduction.

Evidence for delocalized anticooperative flash induced proton binding as revealed by mutants at the M266His iron ligand in bacterial reaction centers.

Bacterial reaction centers (RCs) convert light energy into chemical free energy via the double reduction and protonation of the secondary quinone electron acceptor, QB, to the dihydroquinone QBH2. Two RC mutants (M266His --> Leu and M266His --> Ala) with a modified ligand of the non-heme iron have been studied by flash-induced absorbance change spectroscopy. No important changes were observed for the rate constants of the first and second electron transfers between the first quinone electron acceptor, QA, and QB. However, in the M266HL mutant a destabilization of approximately 40 meV of the free energy level of QA- was observed, at variance with the M266HA mutant. The superposition of the three-dimensional X-ray structures of the three proteins in the QA region provides no obvious explanation for the energy modification in the M266HL mutant. The shift of the midpoint redox potential of QA/QA- in M266HL caused accelerated recombination of the charges in the P+ QA- state of the RCs where the native QA was replaced by a low potential anthraquinone (AQA). As previously reported for the native RCs, in the M266HL we observed a biphasicity of the P+ AQA- --> P AQA charge recombination. Interestingly, both phases present a similar acceleration in the M266HL mutant with respect to the wild type. The pH dependencies of the proton uptake upon QA- and QB- formations are superimposable in both mutants but very different from those of native RCs. The data measured in mutants are similar to those that we previously obtained on strains modified at various sites of the cytoplasmic region. The similarity of the response to these different mutations is puzzling, and we propose that it arises from a collective behavior of multiple acidic residues resulting in strongly anticooperative proton binding. The unspecific disappearance of the high pH band of proton uptake observed in all these mutants appears as the natural consequence of removing any member of an interactive proton cluster. This long range interaction also accounts for the similar responses to mutations of the proton uptake pattern induced by either QA- or QB-. We surmise that the presence of an extended protonated water H-bond network providing protons to QB is responsible for these effects.

Preliminary electrochemical studies of the flavohaemoprotein from Ralstonia eutropha entrapped in a film of methyl cellulose: Activation of the reduction of dioxygen.

A flavohaemoprotein (FHP) from Ralstonia eutropha, obtained in a pure and active form, has been entrapped in a film of methyl cellulose on the electrode surface and gives a stable and reproducible electrochemical response at pH 7.00 when subject to cyclic voltammetry using a glassy carbon electrode. To our knowledge, no previous direct electrochemistry had been achieved with a bacterial flavohaemoglobin, which possess both a FAD and a haem. A single couple is observed which is assigned to the haem moiety of the protein, since the same result is obtained with a semi-apo form of the protein deprived of FAD (semi-apo FHP). The data collected were further confirmed by potentiometry with a platinum electrode, and the homogeneous electron transfer rate estimated by double potential step chronocoulometry at a bare glassy carbon electrode in the presence of methyl viologen (MV). The presence of FAD in the holoprotein is easily confirmed by UV-Vis spectrophotometry, but its expected electron relay role remains elusive. The protein activates the reduction of dioxygen by about 400 mV, the reduction current being proportional to the concentration of dioxygen up to 10% in volume in the gas mixture.