Atomic resolution modeling of the ferredoxin:[FeFe] hydrogenase complex from Chlamydomonas reinhardtii

The [FeFe] hydrogenases HydA1 and HydA2 in the green alga Chlamydomonas reinhardtii catalyze the final reaction in a remarkable metabolic pathway allowing this photosynthetic organism to produce H(2) from water in the chloroplast. A [2Fe-2S] ferredoxin is a critical branch point in electron flow from Photosystem I toward a variety of metabolic fates, including proton reduction by hydrogenases. To better understand the binding determinants involved in ferredoxin:hydrogenase interactions, we have modeled Chlamydomonas PetF1 and HydA2 based on amino-acid sequence homology, and produced two promising electron-transfer model complexes by computational docking. To characterize these models, quantitative free energy calculations at atomic resolution were carried out, and detailed analysis of the interprotein interactions undertaken. The protein complex model we propose for ferredoxin:HydA2 interaction is energetically favored over the alternative candidate by 20 kcal/mol. This proposed model of the electron-transfer complex between PetF1 and HydA2 permits a more detailed view of the molecular events leading up to H(2) evolution, and suggests potential mutagenic strategies to modulate electron flow to HydA2.     

Rational redesign of the ferredoxin-NADP+-oxido-reductase/ferredoxin-interaction for photosynthesis-dependent H2-production

Utilization of electrons from the photosynthetic water splitting reaction for the generation of biofuels, commodities as well as application in biotransformations requires a partial rerouting of the photosynthetic electron transport chain. Due to its rather negative redox potential and its bifurcational function, ferredoxin at the acceptor side of Photosystem 1 is one of the focal points for such an engineering. With hydrogen production as model system, we show here the impact and potential of redox partner design involving ferredoxin (Fd), ferredoxin-oxido-reductase (FNR) and [FeFe]?hydrogenase HydA1 on electron transport in a future cyanobacterial design cell of Synechocystis PCC 6803. X-ray-structure-based rational design and the allocation of specific interaction residues by NMR-analysis led to the construction of Fd- and FNR-mutants, which in appropriate combination enabled an about 18-fold enhanced electron flow from Fd to HydA1 (in competition with equimolar amounts of FNR) in in vitro assays. The negative impact of these mutations on the Fd-FNR electron transport which indirectly facilitates H₂ production (with a contribution of ≤42% by FNR variants and ≤23% by Fd-variants) and the direct positive impact on the Fd-HydA1 electron transport (≤23% by Fd-mutants) provide an excellent basis for the construction of a hydrogen-producing design cell and the study of photosynthetic efficiency-optimization with cyanobacteria.     

Chromosome locations of five genes encoding photosynthetic electron transfer proteins in Arabidopsis thaliana

The chromosome locations of nuclear genes encoding four photosynthetic electron transfer proteins have been determined by examining restriction fragment length polymorphisms in F₈recombinant inbred lines of Arabidopsis thaliana. The single-copy PetC gene encoding the chloroplast Rieske FeS protein was mapped to the top of chromosome 4, whereas the PetE and PetF genes encoding plastocyanin and ferredoxin, respectively, were mapped to different parts of chromosome 1. Two PetH genes encoding ferredoxin-NADP⁺oxidoreductase were mapped to the top of chromosome 1 and the bottom of chromosome 5.     

Electrochemical Studies of a Truncated Laccase Produced in Pichia pastoris

The cDNA that encodes an isoform of laccase from Trametes versicolor (LCCI), as well as a truncated version (LCCIa), was subcloned and expressed by using the yeast Pichia pastoris as the heterologous host. The amino acid sequence of LCCIa is identical to that of LCCI except that the final 11 amino acids at the C terminus of LCCI are replaced with a single cysteine residue. This modification was introduced for the purpose of improving the kinetics of electron transfer between an electrode and the copper-containing active site of laccase. The two laccases (LCCI and LCCIa) are compared in terms of their relative activity with two substrates that have different redox potentials. Results from electrochemical studies on solutions containing LCCI and LCCIa indicate that the redox potential of the active site of LCCIa is shifted to more negative values (411 mV versus normal hydrogen electrode voltage) than that found in other fungal laccases. In addition, replacing the 11 codons at the C terminus of the laccase gene with a single cysteine codon (i.e., LCCI→LCCIa) influences the rate of heterogeneous electron transfer between an electrode and the copper-containing active site (k_het for LCCIa = 1.3 × 10⁻⁴ cm s⁻¹). These results demonstrate for the first time that the rate of electron transfer between an oxidoreductase and an electrode can be enhanced by changes to the primary structure of a protein via site-directed mutagenesis.     

Electron transfer between the spinach plastocyanin mutant Leu12His and Photosystem 1

A spinach plastocyanin (Pc) mutant, Pc(Leu12His), has been constructed by site-directed mutagenesis and expressed in Escherichia coli to probe the importance of the hydrophobic patch in the interaction with Photosystem 1. The mutant has been characterized by optical absorption, EPR spectroscopy and redox titration. The electron transfer to Photosystem 1 was investigated by flash-induced time-resolved absorption measurements at 830 nm. The Pc(Leu12His) mutant showed a major change in the Photosystem 1 kinetics compared to wild-type Pc. In contrast to the biphasic Photosystem 1 reduction observed for the physiological reaction partner, only the slow phase was discerned when Pc(Leu12His) replaced wild-type Pc as the electron donor. The reaction showed a hyperbolic dependence with increasing Pc concentration, saturating at a rate constant value of 2000 s^-1, which is about 10 times slower than the corresponding rate constant for wild-type Pc. Obviously, this position i s critical for a proper reaction. Moreover, the reaction showed a titrating behavior with a pK_a of 6.7. Thus, it appears that both shape and charge of the residue in this position are important. A plausible reaction mechanism for electron transfer between wild-type Pc and Photosystem 1 is discussed. The role of electrostatic interactions may be that of long-range guidance and initial recognition that allow the two proteins to seek a pre-docking configuration(s). Then a short-range rearrangement(s), involving also hydrophobic interactions, forms an optimum docking configuration prior to electron transfer.     

Optimized expression and purification for high-activity preparations of algal [FeFe]-hydrogenase

Background

Recombinant expression and purification of metallo-enzymes, including hydrogenases, at high-yields is challenging due to complex, and enzyme specific, post-translational maturation processes. Low fidelities of maturation result in preparations containing a significant fraction of inactive, apo-protein that are not suitable for biophysical or crystallographic studies.

Principal Findings

We describe the construction, overexpression and high-yield purification of a fusion protein consisting of the algal [2Fe2S]-ferredoxin PetF (Fd) and [FeFe]-hydrogenase HydA1. The maturation of Fd-HydA1 was optimized through improvements in culture conditions and media components used for expression. We also demonstrated that fusion of Fd to the N-terminus of HydA1, in comparison to the C-terminus, led to increased expression levels that were 4-fold higher. Together, these improvements led to enhanced HydA1 activity and improved yield after purification. The strong binding-affinity of Fd for DEAE allowed for two-step purification by ion exchange and StrepTactin affinity chromatography. In addition, the incorporation of a TEV protease site in the Fd-HydA1 linker allowed for the proteolytic removal of Fd after DEAE step, and purification of HydA1 alone by StrepTactin. In combination, this process resulted in HydA1 purification yields of 5 mg L⁻¹ of culture from E. coli with specific activities of 1000 U (U = 1 µmol hydrogen evolved mg⁻¹ min⁻¹).

Significance

The [FeFe]-hydrogenases are highly efficient enzymes and their catalytic sites provide model structures for synthetic efforts to develop robust hydrogen activation catalysts. In order to characterize their structure-function properties in greater detail, and to use hydrogenases for biotechnological applications, reliable methods for rapid, high-yield expression and purification are required.     

Picosecond spectroscopy of the charge separation in reaction centers of Chloroflexus aurantiacus with selective excitation of the primary electron donor

Absorbance changes in the picosecond region were studied in isolated reaction centers of the green photosynthetic bacterium Chloroflexus aurantiacus upon selective excitation of the primary electron donor, P, at 870 nm. The results indicate that the first observed state is an excited state of P (P1) which appears to transfer an electron to a bacteriochlorophyll a molecule absorbing at 812 nm (B₁) in 10 ± 2 ps as indicated by a bleaching at this wavelength. This reaction is followed by a rapid electron transfer (3 ± 1 ps) from B₁⁻ to bacteriopheophytin a, so that the fraction of reaction centers in the state P⁺B₁⁻ remains small during the experiment. An apparent bleaching at 925 nm is ascribed to stimulated emission from excited P, which emission disappears upon formation of P⁺. The difference between these time constants for electron transfer and those observed for the same reactions in reaction centers of the purple photosynthetic bacterium Rhodopseudomonas (Rhodobacter) sphaeroides is discussed in terms of the energy difference between P1 and P⁺B₁⁻, which appears to be larger for C. aurantiacus.     

Pivotal role of the strictly conserved aromatic residue F15 in the cytochrome c 7 family

Cytochromes c ₇ are periplasmic triheme proteins that have been reported exclusively in δ-proteobacteria. The structures of five triheme cytochromes identified in Geobacter sulfurreducens and one in Desulfuromonas acetoxidans have been determined. In addition to the hemes and axial histidines, a single aromatic residue is conserved in all these proteins—phenylalanine 15 (F15). PpcA is a member of the G. sulfurreducens cytochrome c ₇ family that performs electron/proton energy transduction in addition to electron transfer that leads to the reduction of extracellular electron acceptors. For the first time we probed the role of the F15 residue in the PpcA functional mechanism, by replacing this residue with the aliphatic leucine by site-directed mutagenesis. The analysis of NMR spectra of both oxidized and reduced forms showed that the heme core and the overall fold of the mutated protein were not affected. However, the analysis of ¹H–¹⁵N heteronuclear single quantum coherence NMR spectra evidenced local rearrangements in the α-helix placed between hemes I and III that lead to structural readjustments in the orientation of heme axial ligands. The detailed thermodynamic characterization of F15L mutant revealed that the reduction potentials are more negative and the redox-Bohr effect is decreased. The redox potential of heme III is most affected. It is of interest that the mutation in F15, located between hemes I and III in PpcA, changes the characteristics of the two hemes differently. Altogether, these modifications disrupt the balance of the global network of cooperativities, preventing the F15L mutant protein from performing a concerted electron/proton transfer.     

Homologous and Heterologous Overexpression in Clostridium acetobutylicum and Characterization of Purified Clostridial and Algal Fe-Only Hydrogenases with High Specific Activities

Clostridium acetobutylicum ATCC 824 was selected for the homologous overexpression of its Fe-only hydrogenase and for the heterologous expressions of the Chlamydomonas reinhardtii and Scenedesmus obliquus HydA1 Fe-only hydrogenases. The three Strep tag II-tagged Fe-only hydrogenases were isolated with high specific activities by two-step column chromatography. The purified algal hydrogenases evolve hydrogen with rates of around 700 μmol H₂ min⁻¹ mg⁻¹, while HydA from C. acetobutylicum (HydA_Ca) shows the highest activity (5,522 μmol H₂ min⁻¹ mg⁻¹) in the direction of hydrogen uptake. Further, kinetic parameters and substrate specificity were reported. An electron paramagnetic resonance (EPR) analysis of the thionin-oxidized HydA_Ca protein indicates a characteristic rhombic EPR signal that is typical for the oxidized H cluster of Fe-only hydrogenases.     

Histidine residue 252 of the Photosystem II D1 polypeptide is involved in a light-induced cross-linking of the polypeptide with the α subunit of cytochrome b-559: study of a site-directed mutant of Synechocystis PCC 6803

Properties of the Photosystem II (PSII) complex were examined in the wild-type (control) strain of the cyanobacterium Synechocystis PCC 6803 and its site-directed mutant D1-His252Leu in which the histidine residue 252 of the D1 polypeptide was replaced by leucine. This mutation caused a severe blockage of electron transfer between the PSII electron acceptors Q_A and Q_B and largely inhibited PSII oxygen evolving activity. Strong illumination induced formation of a D1-cytochrome b-559 adduct in isolated, detergent-solubilized thylakoid membranes from the control but not the mutant strain. The light-induced generation of the adduct was suppressed after prior modification of thylakoid proteins either with the histidine modifier platinum-terpyridine-chloride or with primary amino group modifiers. Anaerobic conditions and the presence of radical scavengers also inhibited the appearance of the adduct. The data suggest that the D1-cytochrome adduct is the product of a reaction between the oxidized residue His²⁵² of the D1 polypeptide and the N-terminal amino group of the cytochrome α subunit. As the rate of the D1 degradation in the control and mutant strains is similar, formation of the adduct does not seem to represent a required intermediary step in the D1 degradation pathway.     

Unification of [FeFe]-hydrogenases into three structural and functional groups

Background[FeFe]-hydrogenases (Hyd) are structurally diverse enzymes that catalyze the reversible oxidation of hydrogen (H₂). Recent biochemical data demonstrate new functional roles for these enzymes, including those that function in electron bifurcation where an exergonic reaction is coupled with an endergonic reaction to drive the reversible oxidation/production of H₂.MethodsTo identify the structural determinants that underpin differences in enzyme functionality, a total of 714 homologous sequences of the catalytic subunit, HydA, were compiled. Bioinformatics approaches informed by biochemical data were then used to characterize differences in inferred quaternary structure, HydA active site protein environment, accessory iron-sulfur clusters in HydA, and regulatory proteins encoded in HydA gene neighborhoods.ResultsHydA homologs were clustered into one of three classification groups, Group 1 (G1), Group 2 (G2), and Group 3 (G3). G1 enzymes were predicted to be monomeric while those in G2 and G3 were predicted to be multimeric and include HydB, HydC (G2/G3) and HydD (G3) subunits. Variation in the HydA active site and accessory iron-sulfur clusters did not vary by group type. Group-specific regulatory genes were identified in the gene neighborhoods of both G2 and G3 Hyd. Analyses of purified G2 and G3 enzymes by mass spectrometry strongly suggest that they are post-translationally modified by phosphorylation.ConclusionsThese results suggest that bifurcation capability is dictated primarily by the presence of both HydB and HydC in Hyd complexes, rather than by variation in HydA.General significanceThis classification scheme provides a framework for future biochemical and mutagenesis studies to elucidate the functional role of Hyd enzymes.     

Evidence from directed mutagenesis that positively charged amino acids are necessary for interaction of nitrogenase with the [2Fe-2S) heterocyst ferredoxin (FdxH) from the cyanobacterium Anabaena sp., PCC7120

Sequence comparison of the heterocyst-type ferredoxin (FdxH) from Anabaena 7120 and type-I ferredoxins (PetF) from the same organism and other cyanobacteria revealed a group of positively charged residues characteristic for FdxH. Molecular modeling showed that these basic amino acids are clustered on the surface of FdxH. The corresponding domain of PetF contained acidic or nonpolar residues instead. To identify amino acids that are important for interaction with nitrogenase, we generated site-directed mutations in the fdxH gene and assayed the in vitro activity of the resulting recombinant proteins isolated from Escherichia coli. In addition to the point mutants, two chimeric proteins, FdxH : PetF and PetF : FdxH, were constructed containing the 58 N-terminal amino acids of one ferredoxin fused to the 40 C-terminal amino acids of the other. Exchange of lysines 10 and 11 of FdxH for the corresponding residues of PetF (glutamate 10 and alanine 11) resulted in a ferredoxin with greatly decreased affinity to nitrogenase. This indicates an important function of these basic amino acids in interaction with dinitrogenase reductase (NifH) from Anabaena. In addition we checked the reactivity of the recombinant ferredoxins with ferredoxin-NADP⁺ oxidoreductase (FNR) and photosystem I. The experiments with both the chimeric and point mutated ferredoxins showed that the C-terminal part of this protein determines its activity in NADP⁺ photoreduction.     

Protein Crystallography Reveals a Role for the FS0 Cluster of Escherichia coli Nitrate Reductase A (NarGHI) in Enzyme Maturation

We have used site-directed mutagenesis, EPR spectroscopy, redox potentiometry, and protein crystallography to monitor assembly of the FS0 [4Fe-4S] cluster and molybdo-bis(pyranopterin guanine dinucleotide) cofactor (Mo-bisPGD) of the Escherichia coli nitrate reductase A (NarGHI) catalytic subunit (NarG). Cys and Ser mutants of NarG-His⁴⁹ both lack catalytic activity, with only the former assembling FS0 and Mo-bisPGD. Importantly, both prosthetic groups are absent in the NarG-H49S mutant. EPR spectroscopy of the Cys mutant reveals that the E_m value of the FS0 cluster is decreased by at least 500 mV, preventing its participation in electron transfer to the Mo-bisPGD cofactor. To demonstrate that decreasing the FS0 cluster E_m results in decreased enzyme activity, we mutated a critical Arg residue (NarG-Arg⁹⁴) in the vicinity of FS0 to a Ser residue. In this case, the E_m of FS0 is decreased by 115 mV, with a concomitant decrease in enzyme turnover to ∼30% of the wild type. Analysis of the structure of the NarG-H49S mutant reveals two important aspects of NarGHI maturation: (i) apomolybdo-NarGHI is able to bind GDP moieties at their respective P and Q sites in the absence of the Mo-bisPGD cofactor, and (ii) a critical segment of residues in NarG, ⁴⁹HGVNCTG⁵⁵, must be correctly positioned to ensure holoenzyme maturation.     

Engineering of dye-mediated dehydrogenase property of fructosyl amino acid oxidases by site-directed mutagenesis studies of its putative proton relay system

The flavoenzyme fructosyl amino acid oxidase (FAOD) catalyzes the oxidative deglycation of fructosyl amino acids, model compounds of glycated proteins. The high oxygen reactivity of FAODs limits their potential utility in amperometric enzyme sensors employing artificial electron mediators. To alter their electron acceptor availability, site-directed mutagenesis was carried out on conserved residues predicted to be involved in the proton relay system (PRS) of two eukaryotic FAODs, the FAOD from the marine yeast Pichia sp. N1-1 and amadoriase II from the fungus Aspergillus fumigatus. The substitution of a single conserved Asn residue in the putative PRS, Asn47Ala of N1-1 FAOD and Asn52Ala of amadoriase II, resulted in significant loss in the catalytic ability to employ O₂ as the electron acceptor, while having little effect on the dye-mediated dehydrogenase activity employing artificial electron acceptors instead of O₂.     

Intraprotein electron transfer in a ruthenium-modified Tyr83-His plastocyanin mutant: evidence for strong electronic coupling

A site-directed mutant of spinach plastocyanin, Pc(Tyr83-His), has been modified by covalent attachment of a photoactive [Ru(bpy)₂(im)]²⁺ complex to the His83 residue. The residue is surface exposed and located about 10–12?Å from the copper ion at the entrance of a proposed natural electron transfer pathway from cytochrome f. Electron transfer within the Ru-Pc complex has been studied with time-resolved optical spectroscopy using two different approaches. In the first, the fully reduced [Cu(I), Ru(II)] protein was photoexcited and subsequently oxidized by an external quencher, forming the [Cu(I), Ru(III)] protein. This was followed by an electron transfer from reduced Cu(I) to Ru(III). In the second method, the initially oxidized Cu(II) ion acted as an internal quencher for excited Ru(II) and the photoinduced reduction of the Cu(II) ion was followed by a thermal recombination with the Ru(III) ion. The reoxidation of the Cu ion, which has an estimated driving force of 0.56?eV, occured with a rate constant k _et?=?(9.5±1.0)×10⁶?s^–1, observed with both methods. The results suggest a strong electronic coupling (H _DA>0.3?cm^–1) along the Ru-His(83)-Cys(84)-Cu pathway.     

Sequential assembly of photosynthetic units in Rhodobacter sphaeroides as revealed by fast repetition rate analysis of variable bacteriochlorophyll a fluorescence

The development of functional photosynthetic units in Rhodobacter sphaeroides was followed by near infra-red fast repetition rate (IRFRR) fluorescence measurements that were correlated to absorption spectroscopy, electron microscopy and pigment analyses. To induce the formation of intracytoplasmic membranes (ICM) (greening), cells grown aerobically both in batch culture and in a carbon-limited chemostat were transferred to semiaerobic conditions. In both aerobic cultures, a low level of photosynthetic complexes was observed, which were composed of the reaction center and the LH1 core antenna. Interestingly, in the batch cultures the reaction centers were essentially inactive in forward electron transfer and exhibited low photochemical yields F_V/F_M, whereas the chemostat culture displayed functional reaction centers with a rather rapid (1-2 ms) electron transfer turnover, as well as a high F_V/F_M of ∼0.8. In both cases, the transfer to semiaerobiosis resulted in rapid induction of bacteriochlorophyll a synthesis that was reflected by both an increase in the number of LH1-reaction center and peripheral LH2 antenna complexes. These studies establish that photosynthetic units are assembled in a sequential manner, where the appearance of the LH1-reaction center cores is followed by the activation of functional electron transfer, and finally by the accumulation of the LH2 complexes.     

Photosystem I light-harvesting proteins regulate photosynthetic electron transfer and hydrogen production

Linear electron flow (LEF) and cyclic electron flow (CEF) compete for light-driven electrons transferred from the acceptor side of photosystem I (PSI). Under anoxic conditions, such highly reducing electrons also could be used for hydrogen (H₂) production via electron transfer between ferredoxin and hydrogenase in the green alga Chlamydomonas reinhardtii. Partitioning between LEF and CEF is regulated through PROTON-GRADIENT REGULATION5 (PGR5). There is evidence that partitioning of electrons also could be mediated via PSI remodeling processes. This plasticity is linked to the dynamics of PSI-associated light-harvesting proteins (LHCAs) LHCA2 and LHCA9. These two unique light-harvesting proteins are distinct from all other LHCAs because they are loosely bound at the PSAL pole. Here, we investigated photosynthetic electron transfer and H₂ production in single, double, and triple mutants deficient in PGR5, LHCA2, and LHCA9. Our data indicate that lhca2 and lhca9 mutants are efficient in photosynthetic electron transfer, that LHCA2 impacts the pgr5 phenotype, and that pgr5/lhca2 is a potent H₂ photo-producer. In addition, pgr5/lhca2 and pgr5/lhca9 mutants displayed substantially different H₂ photo-production kinetics. This indicates that the absence of LHCA2 or LHCA9 impacts H₂ photo-production independently, despite both being attached at the PSAL pole, pointing to distinct regulatory capacities.

Alteration of the light-harvesting composition of photosystem I impacts photosynthetic electron transfer and hydrogen production.     

Functional and fluorescence analyses of tryptophan residues in H+-pyrophosphatase of Clostridium tetani

Homodimeric proton-translocating pyrophosphatase (H⁺-PPase; EC 3.6.1.1) maintains the cytoplasmic pH homeostasis of many bacteria and higher plants by coupling pyrophosphate (PP_i) hydrolysis and proton translocation. H⁺-PPase accommodates several essential motifs involved in the catalytic mechanism, including the PP_i binding motif and Acidic I and II motifs. In this study, 3 intrinsic tryptophan residues, Trp-75, Trp-365, and Trp-602, in H⁺-PPase from Clostridium tetani were used as internal probes to monitor the local conformational state of the periplasm domain, transmembrane region, and cytoplasmic domain, respectively. Upon binding of the substrate analog Mg-imidodiphosphate (Mg-IDP), local structural changes prevented the modification of tryptophan residues by N-bromosuccinimide (NBS), especially at Trp-602. Following Mg-P_i binding, Trp-75 and Trp-365, but not Trp-602, were slightly protected from structural modifications by NBS. These results reveal the conformation of H⁺-PPase is distinct in the presence of different ligands. Moreover, analyses of the Stern-Volmer relationship and steady-state fluorescence anisotropy also indicate that the local structure around Trp-602 is more exposed to solvent and varied under different environments. In addition, Trp-602 was identified to be a crucial residue in the H⁺-PPase that may potentially be involved in stabilizing the structure of the catalytic region by site-directed mutagenesis analysis.