RESUMEN
The photo-induced oxidation of TyrZ and TyrD by P680(â¢+), that involves both electron and proton transfer (PCET), has been studied in oxygen-evolving photosystem II from Thermosynechococcus elongatus. We used time-resolved absorption spectroscopy to measure the kinetics of P680(â¢+) reduction by tyrosine after the first flash given to dark-adapted PS II as a function of temperature and pH. The half-life of TyrZ oxidation by P680(â¢+) increases from 20ns at 300K to about 4µs at 150K. Analyzing the temperature dependence of the rate, one obtains a reorganization energy of about 770meV. Between 260K and 150K, the reduction of P680(â¢+) by TyrZ is increasingly replaced by charge recombination between P680(â¢+) and QA(â¢-). We propose that the driving force for TyrZ oxidation by P680(â¢+) decreases upon lowering the temperature. TyrZ oxidation cannot be excluded in a minority of PS II complexes at cryogenic temperatures. TyrD oxidation by P680(â¢+) with a half-life of about 30ns was observed at high pH. The pH dependence of the yield of TyrD oxidation can be described by a single protonable group with a pK of approximately 8.4. The rate of TyrD oxidation by P680(â¢+) is virtually identical upon substitution of solvent exchangeable protons with deuterons indicating that the rate is limited by electron transfer. The rate is independent of temperature between 5K and 250K. It is concluded that TyrD donates the electron to P680(â¢+) via PD2.
RESUMEN
Hydrogenases catalyze the reversible oxidation of H(2) into protons and electrons and are usually readily inactivated by O(2). However, a subgroup of the [NiFe] hydrogenases, including the membrane-bound [NiFe] hydrogenase from Ralstonia eutropha, has evolved remarkable tolerance toward O(2) that enables their host organisms to utilize H(2) as an energy source at high O(2). This feature is crucially based on a unique six cysteine-coordinated [4Fe-3S] cluster located close to the catalytic center, whose properties were investigated in this study using a multidisciplinary approach. The [4Fe-3S] cluster undergoes redox-dependent reversible transformations, namely iron swapping between a sulfide and a peptide amide N. Moreover, our investigations unraveled the redox-dependent and reversible occurence of an oxygen ligand located at a different iron. This ligand is hydrogen bonded to a conserved histidine that is essential for H(2) oxidation at high O(2). We propose that these transformations, reminiscent of those of the P-cluster of nitrogenase, enable the consecutive transfer of two electrons within a physiological potential range.
Asunto(s)
Hidrogenasas/metabolismo , Proteínas Hierro-Azufre/metabolismo , Oxígeno/metabolismo , Catálisis , Hidrógeno/metabolismo , Ligandos , Modelos Moleculares , Oxidación-ReducciónRESUMEN
The soluble NAD(+)-reducing hydrogenase (SH) from Ralstonia eutropha H16 belongs to the O2-tolerant subtype of pyridine nucleotide-dependent [NiFe]-hydrogenases. To identify molecular determinants for the O2 tolerance of this enzyme, we introduced single amino acids exchanges in the SH small hydrogenase subunit. The resulting mutant strains and proteins were investigated with respect to their physiological, biochemical, and spectroscopic properties. Replacement of the four invariant conserved cysteine residues, Cys41, Cys44, Cys113, and Cys179, led to unstable protein, strongly supporting their involvement in the coordination of the iron-sulfur cluster proximal to the catalytic [NiFe] center. The Cys41Ser exchange, however, resulted in an SH variant that displayed up to 10% of wild-type activity, suggesting that the coordinating role of Cys41 might be partly substituted by the nearby Cys39 residue, which is present only in O2-tolerant pyridine nucleotide-dependent [NiFe]-hydrogenases. Indeed, SH variants carrying glycine, alanine, or serine in place of Cys39 showed increased O2 sensitivity compared to that of the wild-type enzyme. Substitution of further amino acids typical for O2-tolerant SH representatives did not greatly affect the H2-oxidizing activity in the presence of O2. Remarkably, all mutant enzymes investigated by electron paramagnetic resonance spectroscopy did not reveal significant spectral changes in relation to wild-type SH, showing that the proximal iron-sulfur cluster does not contribute to the wild-type spectrum. Interestingly, exchange of Trp42 by serine resulted in a completely redox-inactive [NiFe] site, as revealed by infrared spectroscopy and H2/D(+) exchange experiments. The possible role of this residue in electron and/or proton transfer is discussed.
Asunto(s)
Cupriavidus necator/enzimología , Hidrogenasas/química , Hidrogenasas/metabolismo , Oxígeno/metabolismo , Sustitución de Aminoácidos , Dominio Catalítico , Cupriavidus necator/química , Cupriavidus necator/genética , Cupriavidus necator/metabolismo , Espectroscopía de Resonancia por Spin del Electrón , Hidrogenasas/genética , Hierro/química , Hierro/metabolismo , Modelos Moleculares , NAD/metabolismo , Espectroscopía Infrarroja por Transformada de Fourier , Azufre/química , Azufre/metabolismoRESUMEN
The membrane-bound [NiFe] hydrogenase (MBH) supports growth of Ralstonia eutropha H16 with H2 as the sole energy source. The enzyme undergoes a complex biosynthesis process that proceeds during cell growth even at ambient O2 levels and involves 14 specific maturation proteins. One of these is a rubredoxin-like protein, which is essential for biosynthesis of active MBH at high oxygen concentrations but dispensable under microaerobic growth conditions. To obtain insights into the function of HoxR, we investigated the MBH protein purified from the cytoplasmic membrane of hoxR mutant cells. Compared with wild-type MBH, the mutant enzyme displayed severely decreased hydrogenase activity. Electron paramagnetic resonance and infrared spectroscopic analyses revealed features resembling those of O2-sensitive [NiFe] hydrogenases and/or oxidatively damaged protein. The catalytic center resided partially in an inactive Niu-A-like state, and the electron transfer chain consisting of three different Fe-S clusters showed marked alterations compared with wild-type enzyme. Purification of HoxR protein from its original host, R. eutropha, revealed only low protein amounts. Therefore, recombinant HoxR protein was isolated from Escherichia coli. Unlike common rubredoxins, the HoxR protein was colorless, rather unstable, and essentially metal-free. Conversion of the atypical iron-binding motif into a canonical one through genetic engineering led to a stable reddish rubredoxin. Remarkably, the modified HoxR protein did not support MBH-dependent growth at high O2. Analysis of MBH-associated protein complexes points toward a specific interaction of HoxR with the Fe-S cluster-bearing small subunit. This supports the previously made notion that HoxR avoids oxidative damage of the metal centers of the MBH, in particular the unprecedented Cys6[4Fe-3S] cluster.
Asunto(s)
Proteínas Bacterianas/química , Hidrogenasas/biosíntesis , Rubredoxinas/química , Catálisis , Membrana Celular/enzimología , Cupriavidus necator/enzimología , Espectroscopía de Resonancia por Spin del Electrón , Transporte de Electrón , Metales/química , Modelos Químicos , Oxidación-Reducción , Oxígeno/química , Plásmidos/metabolismo , Espectrofotometría Ultravioleta , Espectroscopía Infrarroja por Transformada de FourierRESUMEN
In Photosystem I (PS I) long-wavelength chlorophylls (LWC) of the core antenna are known to extend the spectral region up to 750 nm for absorbance of light that drives photochemistry. Here we present clear evidence that even far-red light with wavelengths beyond 800 nm, clearly outside the LWC absorption bands, can still induce photochemical charge separation in PS I throughout the full temperature range from 295 to 5 K. At room temperature, the photoaccumulation of P700(+â¢) was followed by the absorbance increase at 826 nm. At low temperatures (T < 100 K), the formation of P700(+â¢)FA/B(-â¢) was monitored by the characteristic EPR signals of P700(+â¢) and FA/B(-â¢) and by the characteristic light-minus-dark absorbance difference spectrum in the QY region. P700 oxidation was observed upon selective excitation at 754, 785, and 808 nm, using monomeric and trimeric PS I core complexes of Thermosynechococcus elongatus and Arthrospira platensis, which differ in the amount of LWC. The results show that the LWC cannot be responsible for the long-wavelength excitation-induced charge separation at low temperatures, where thermal uphill energy transfer is frozen out. Direct energy conversion of the excitation energy from the LWC to the primary radical pair, e.g., via a superexchange mechanism, is excluded, because no dependence on the content of LWC was observed. Therefore, it is concluded that electron transfer through PS I is induced by direct excitation of a proposed charge transfer (CT) state in the reaction center. A direct signature of this CT state is seen in absorbance spectra of concentrated PS I samples, which reveal a weak and featureless absorbance band extending beyond 800 nm, in addition to the well-known bands of LWC (C708, C719 and C740) in the range between 700 and 750 nm. The present findings suggest that nature can exploit CT states for extending the long wavelength limit in PSI even beyond that of LWC. Similar mechanisms may work in other photosynthetic systems and in chemical systems capable of photoinduced electron transfer processes in general.
Asunto(s)
Proteínas Bacterianas/química , Clorofila/química , Cianobacterias/química , Complejo de Proteína del Fotosistema I/química , Proteínas Bacterianas/metabolismo , Clorofila/metabolismo , Cianobacterias/metabolismo , Transporte de Electrón , Luz , Modelos Moleculares , Oxidación-Reducción , Procesos Fotoquímicos , Complejo de Proteína del Fotosistema I/metabolismo , TemperaturaRESUMEN
Hydrogenases are essential for H(2) cycling in microbial metabolism and serve as valuable blueprints for H(2)-based biotechnological applications. However, most hydrogenases are extremely oxygen sensitive and prone to inactivation by even traces of O(2). The O(2)-tolerant membrane-bound [NiFe]-hydrogenase of Ralstonia eutropha H16 is one of the few examples that can perform H(2) uptake in the presence of ambient O(2). Here we show that O(2) tolerance is crucially related to a modification of the internal electron-transfer chain. The iron-sulfur cluster proximal to the active site is surrounded by six instead of four conserved coordinating cysteines. Removal of the two additional cysteines alters the electronic structure of the proximal iron-sulfur cluster and renders the catalytic activity sensitive to O(2) as shown by physiological, biochemical, spectroscopic and electrochemical studies. The data indicate that the mechanism of O(2) tolerance relies on the reductive removal of oxygenic species guided by the unique architecture of the electron relay rather than a restricted access of O(2) to the active site.
Asunto(s)
Cupriavidus necator/metabolismo , Hidrogenasas/metabolismo , Proteínas Hierro-Azufre/metabolismo , Biocatálisis , Dióxido de Carbono/química , Dióxido de Carbono/metabolismo , Dominio Catalítico , Cupriavidus necator/enzimología , Cisteína/química , Cisteína/metabolismo , Electroquímica , Electroforesis en Gel de Poliacrilamida , Hidrógeno/química , Hidrógeno/metabolismo , Hidrogenasas/química , Proteínas Hierro-Azufre/química , Oxígeno/química , Oxígeno/metabolismo , Conformación Proteica , Espectroscopía Infrarroja por Transformada de FourierRESUMEN
Activation of the corrinoid [Fe-S] protein (CoFeSP), involved in reductive CO(2) conversion, requires the reduction of the Co(II) center by the [Fe-S] protein RACo, which according to the reduction potentials of the two proteins would correspond to an uphill electron transfer. In our resonance Raman spectroscopic work, we demonstrate that, as a conformational gate for the corrinoid reduction, complex formation of Co(II)FeSP and RACo specifically alters the structure of the corrinoid cofactor by modifying the interactions of the Co(II) center with the axial ligand. On the basis of various deletion mutants, the potential interaction domains on the partner proteins can be predicted.
Asunto(s)
Corrinoides/química , Corrinoides/metabolismo , Proteínas Hierro-Azufre/química , Proteínas Hierro-Azufre/metabolismo , Espectroscopía de Resonancia por Spin del Electrón , Unión Proteica , Espectrometría RamanRESUMEN
Dark operative protochlorophyllide oxidoreductase (DPOR) catalyzes the light-independent two-electron reduction of protochlorophyllide a to form chlorophyllide a, the last common precursor of chlorophyll a and bacteriochlorophyll a biosynthesis. During ATP-dependent DPOR catalysis the homodimeric ChlL(2) subunit carrying a [4Fe-4S] cluster transfers electrons to the corresponding heterotetrameric catalytic subunit (ChlN/ChlB)(2), which also possesses a redox active [4Fe-4S] cluster. To investigate the transient interaction of both subcomplexes and the resulting electron transfer reactions, the ternary DPOR enzyme holocomplex comprising subunits ChlN, ChlB, and ChlL from the cyanobacterium Prochlorococcus marinus was trapped as an octameric (ChlN/ChlB)(2)(ChlL(2))(2) complex after incubation with the nonhydrolyzable ATP analogs adenosine 5'-(gamma-thio)triphosphate, adenosine 5'-(beta,gamma-imido)triphosphate, or MgADP in combination with AlF(4)(-). Additionally, a mutant ChlL(2) protein, with a deleted Leu(153) in the switch II region also allowed for the formation of a stable octameric complex. Furthermore, efficient complex formation required the presence of protochlorophyllide. Electron paramagnetic resonance spectroscopy of ternary DPOR complexes revealed a reduced [4Fe-4S] cluster located on ChlL(2), indicating that complete ATP hydrolysis is a prerequisite for intersubunit electron transfer. Circular dichroism spectroscopic experiments indicated nucleotide-dependent conformational changes for ChlL(2) after ATP binding. A nucleotide-dependent switch mechanism triggering ternary complex formation and electron transfer was concluded. From these results a detailed redox cycle for DPOR catalysis was deduced.
Asunto(s)
Adenosina Trifosfato/metabolismo , Bacterioclorofilas/biosíntesis , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/metabolismo , Fotosíntesis/fisiología , Prochlorococcus/enzimología , Catálisis , Dicroismo Circular , Espectroscopía de Resonancia por Spin del Electrón , Transporte de Electrón/fisiología , Hierro/metabolismo , Mutagénesis Sitio-Dirigida , Oxidación-Reducción , Subunidades de Proteína/metabolismo , Azufre/metabolismoRESUMEN
Aldehyde oxidase (AOX) is characterized by a broad substrate specificity, oxidizing aromatic azaheterocycles, such as N¹-methylnicotinamide and N-methylphthalazinium, or aldehydes, such as benzaldehyde, retinal, and vanillin. In the past decade, AOX has been recognized increasingly to play an important role in the metabolism of drugs through its complex cofactor content, tissue distribution, and substrate recognition. In humans, only one AOX gene (AOX1) is present, but in mouse and other mammals different AOX homologs were identified. The multiple AOX isoforms are expressed tissue-specifically in different organisms, and it is believed that they recognize distinct substrates and carry out different physiological tasks. AOX is a dimer with a molecular mass of approximately 300 kDa, and each subunit of the homodimeric enzyme contains four different cofactors: the molybdenum cofactor, two distinct [2Fe-2S] clusters, and one FAD. We purified the AOX homolog from mouse liver (mAOX3) and established a system for the heterologous expression of mAOX3 in Escherichia coli. The purified enzymes were compared. Both proteins show the same characteristics and catalytic properties, with the difference that the recombinant protein was expressed and purified in a 30% active form, whereas the native protein is 100% active. Spectroscopic characterization showed that FeSII is not assembled completely in mAOX3. In addition, both proteins were crystallized. The best crystals were from native mAOX3 and diffracted beyond 2.9 Å. The crystals belong to space group P1, and two dimers are present in the unit cell.
Asunto(s)
Aldehído Oxidorreductasas/química , Aldehído Oxidorreductasas/metabolismo , Escherichia coli/enzimología , Hígado/enzimología , Aldehído Oxidorreductasas/genética , Animales , Dicroismo Circular/métodos , Cristalización/métodos , Espectroscopía de Resonancia por Spin del Electrón/métodos , Escherichia coli/genética , Cinética , Ratones , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidad por SustratoRESUMEN
The bidirectional [NiFe] hydrogenase of the cyanobacterium Synechocystis sp. PCC 6803 was purified to apparent homogeneity by a single affinity chromatography step using a Synechocystis mutant with a Strep-tag II fused to the C terminus of HoxF. To increase the yield of purified enzyme and to test its overexpression capacity in Synechocystis the psbAII promoter was inserted upstream of the hoxE gene. In addition, the accessory genes (hypF, C, D, E, A, and B) from Nostoc sp. PCC 7120 were expressed under control of the psbAII promoter. The respective strains show higher hydrogenase activities compared with the wild type. For the first time a Fourier transform infrared (FTIR) spectroscopic characterization of a [NiFe] hydrogenase from an oxygenic phototroph is presented, revealing that two cyanides and one carbon monoxide coordinate the iron of the active site. At least four different redox states of the active site were detected during the reversible activation/inactivation. Although these states appear similar to those observed in standard [NiFe] hydrogenases, no paramagnetic nickel state could be detected in the fully oxidized and reduced forms. Electron paramagnetic resonance spectroscopy confirms the presence of several iron-sulfur clusters after reductive activation. One [4Fe4S](+) and at least one [2Fe2S](+) cluster could be identified. Catalytic amounts of NADH or NADPH are sufficient to activate the reaction of this enzyme with hydrogen.
Asunto(s)
Proteínas Bacterianas/química , Hidrogenasas/química , Hierro/química , NADP/química , Níquel/química , Synechocystis/enzimología , Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/genética , Proteínas Bacterianas/aislamiento & purificación , Espectroscopía de Resonancia por Spin del Electrón , Activación Enzimática , Expresión Génica , Hidrogenasas/biosíntesis , Hidrogenasas/genética , Hidrogenasas/aislamiento & purificación , Nostoc/enzimología , Nostoc/genética , Oxidación-Reducción , Espectroscopía Infrarroja por Transformada de Fourier/métodos , Synechocystis/genéticaRESUMEN
The chemistry of bacterial photosynthesis begins in the photosynthetic reaction centre (RC), a protein complex containing a series of electron donor and acceptor molecules. Although the pigments of the RC can absorb light to operate the photochemistry, the bulk of the light is captured in special pigmented proteins, the light harvesting complexes (LHCs), that then transfer the energy to the RC. Ordinarily, the LHCs do not participate in chemical reactions during photosynthesis such that LHCs do not become oxidised upon light irradiation. However, upon chemical oxidation in the dark, cation radicals of bacteriochlorophyll (BChl) can be formed in the light harvesting complex 1 (LH1) of Rhodobacter sphaeroides. As observed by continuous-wave electron-paramagnetic resonance (EPR), the charges of the BChl(+) cations migrate rather freely about the LH1 complex as in a molecular wire. Remarkably, these LH1 molecular wires continue to function in the frozen, solid state. To investigate the nature of electron-hole transfer and to corroborate the process as revealed by EPR, electron-nuclear double resonance (ENDOR) was recorded at 80 K. ENDOR observed only monomeric bacteriochlorophyll cations. Their signal intensity decreased with increased oxidation while the EPR signal narrowed and increased in size. At the increased oxidation state, the possibility of spin-spin exchange between two BChl(+)s within LH1 versus electron-hole transfer is addressed. An energy landscape of the BChl(+)s in the LH1 is proposed to explain the EPR and ENDOR results.
Asunto(s)
Espectroscopía de Resonancia por Spin del Electrón , Complejos de Proteína Captadores de Luz/química , Rhodobacter sphaeroides/enzimología , Transporte de Electrón , Oxidación-ReducciónRESUMEN
[NiFe] hydrogenases are widespread among microorganisms and catalyze the reversible cleavage of molecular hydrogen. However, only a few bacteria, such as Ralstonia eutropha H16 (Re), synthesize [NiFe] hydrogenases that perform H(2) cycling in the presence of O(2). These enzymes are of special interest for biotechnological applications. To gain further insight into the mechanism(s) responsible for the remarkable O(2) tolerance, we employ FTIR and EPR spectroscopy to study mutant variants of the membrane-bound hydrogenase (MBH) of Re-carrying substitutions of a particular cysteine residue in the vicinity of the [NiFe] active site that is characteristic of O(2)-tolerant membrane-bound [NiFe] hydrogenases. We demonstrate that these MBH variants, despite minor changes in the electronic structure and in the interaction behavior with the embedding protein matrix, display all relevant catalytic and noncatalytic states of the wild-type enzyme, as long as they are still located in the cytoplasmic membrane. Notably, in the oxidized Ni(r)-B state and the fully reduced forms, the CO stretching frequency increases with increasing polarity of the respective amino acid residue at the specific position of the cysteine residue. We purified the MBH mutant protein with a cysteine-to-alanine exchange to apparent homogeneity as dimeric enzyme after detergent solubilization from the membrane. This purified version displays increased oxygen sensitivity, which is reflected by detection of the oxygen-inhibited Ni(u)-A state, an irreversible inactive redox state, and the light-induced Ni(a)-L state even at room temperature.
Asunto(s)
Hidrogenasas/química , Oxígeno/química , Sustitución de Aminoácidos , Dominio Catalítico , Cupriavidus necator/enzimología , Espectroscopía de Resonancia por Spin del Electrón , Hidrogenasas/genética , Mutagénesis Sitio-Dirigida , Estructura Terciaria de Proteína , Espectroscopía Infrarroja por Transformada de FourierRESUMEN
The geometric and electronic structures of the active sites in the oxidized Ni(r)-B state of the [NiFe] hydrogenases from Ralstonia eutropha H16 and Desulfovibrio vulgaris Miyazaki F were investigated in pulsed EPR and ENDOR experiments at two different microwave frequencies (X- and Q-band). Two hyperfine-couplings were clearly resolved in the frozen solution spectra arising from the beta-protons of the nickel-coordinating cysteine residues Cys549 and Cys586 from the Desulfovibrio vulgaris and Ralstonia eutropha hydrogenase, respectively. ESEEM spectroscopic experiments reveal the presence of a histidine in the second coordination sphere of the Ni. The spectroscopic data indicate that the electronic structures of the [NiFe] centers in both hydrogenases are identical in the Ni(r)-B state. However, additional spin couplings of the active site to further paramagnetic centers were identified for the Ralstonia eutropha hydrogenase. The respective couplings could be clearly resolved and simulated. The results from this study are discussed in view of the exceptional O(2)-tolerance of the Ralstonia hydrogenase.
Asunto(s)
Cupriavidus necator/enzimología , Desulfovibrio vulgaris/enzimología , Hidrogenasas/química , Dominio Catalítico , Espectroscopía de Resonancia por Spin del Electrón , Enlace de Hidrógeno , Oxidación-Reducción , Marcadores de SpinRESUMEN
A recently discovered subgroup of class I ribonucleotide reductase (RNR) found in the infectious bacterium Chlamydia trachomatis (C. trachomatis) was shown to exhibit a high-valent Fe(III)Fe(IV) center instead of the tyrosyl radical observed normally in all class I RNRs. The X-ray structure showed that C. trachomatis WT RNR has a phenylalanine at the position of the active tyrosine in Escherichia coli RNR. In this paper the X-ray structure of variant F127Y is presented, where the tyrosine is restored. Using (1)H- and (57)Fe-ENDOR spectroscopy it is shown, that in WT and variants F127Y and Y129F of C. trachomatis RNR, the Fe(III)Fe(IV) center is virtually identical with the short-lived intermediate X observed during the iron oxygen reconstitution reaction in class I RNR from E. coli. The experimental data are consistent with a recent theoretical model for X, proposing two bridging oxo ligands and one terminal water ligand. A surprising extension of the lifetime of the Fe(III)Fe(IV) state in C. trachomatis from a few seconds to several hours at room temperature was observed under catalytic conditions in the presence of substrate. These findings suggest a possible new role for the Fe(III)Fe(IV) state also in other class I RNR, during the catalytic radical transfer reaction, by which the substrate turnover is started.
Asunto(s)
Chlamydia trachomatis/enzimología , Compuestos Férricos/química , Compuestos Ferrosos/química , Ribonucleótido Reductasas/química , Sustitución de Aminoácidos/genética , Chlamydia trachomatis/genética , Cristalografía por Rayos X , Espectroscopía de Resonancia por Spin del Electrón , Mutación Puntual , Ribonucleótido Reductasas/genética , Especificidad por Sustrato/genéticaRESUMEN
Ribonucleotide reductase (class I) contains two components: protein R1 binds the substrate, and protein R2 normally has a diferric site and a tyrosyl free radical needed for catalysis. In Chlamydia trachomatis RNR, protein R2 functions without radical. Enzyme activity studies show that in addition to a diiron cluster, a mixed manganese-iron cluster provides the oxidation equivalent needed to initiate catalysis. An EPR signal was observed from an antiferromagnetically coupled high-spin Mn(III)-Fe(III) cluster in a catalytic reaction mixture with added inhibitor hydroxyurea. The manganese-iron cluster in protein R2 confers much higher specific activity than the diiron cluster does to the enzyme.
Asunto(s)
Chlamydia trachomatis/enzimología , Hierro/química , Hierro/metabolismo , Manganeso/química , Manganeso/metabolismo , Ribonucleótido Reductasas/química , Ribonucleótido Reductasas/metabolismo , Catálisis , Chlamydia trachomatis/genética , Espectroscopía de Resonancia por Spin del Electrón , Ribonucleótido Reductasas/genéticaRESUMEN
This short review compiles high-field electron paramagnetic resonance (EPR) and electron nuclear double resonance (ENDOR) studies on different intermediate amino acid radicals, which emerge in wild-type and mutant class I ribonucleotide reductase (RNR) both in the reaction of protein subunit R2 with molecular oxygen, which generates the essential tyrosyl radical, and in the catalytic reaction, which involves a radical transfer between subunits R2 and R1. Recent examples are presented, how different amino acid radicals (tyrosyl, tryptophan, and different cysteine-based radicals) were identified, assigned to a specific residue, and their interactions, in particular hydrogen bonding, were investigated using high-field EPR and ENDOR spectroscopy. Thereby, unexpected diiron-radical centers, which emerge in mutants of R2 with changed iron coordination, and an important catalytic cysteine-based intermediate in the substrate turnover reaction in R1 were identified and characterized. Experiments on the essential tyrosyl radical in R2 single crystals revealed the so far unknown conformational changes induced by formation of the radical. Interesting structural differences between the tyrosyl radicals of class Ia and Ib enzymes were revealed. Recently accurate distances between the tyrosyl radicals in the protein dimer R2 could be determined using pulsed electron-electron double resonance (PELDOR), providing a new tool for docking studies of protein subunits. These studies show that high-field EPR and ENDOR are important tools for the identification and investigation of radical intermediates, which contributed significantly to the current understanding of the reaction mechanism of class I RNR.
Asunto(s)
Aminoácidos/química , Radicales Libres/química , Modelos Químicos , Ribonucleótido Reductasas/química , Catálisis , Espectroscopía de Resonancia por Spin del Electrón/métodos , Oxígeno/química , Conformación Proteica , Subunidades de Proteína/químicaRESUMEN
The triplet states in plant photosystem II (PS II), 3P680, and from chlorophyll a, 3Chl a, in organic solution have been investigated using pulse ENDOR combined with repetitive laser excitation at cryogenic temperature with the aim to obtain their hyperfine (hf) structure. The large zero field splitting (ZFS) tensor of 3P680 enabled orientation selection via the electron spin resonance (EPR) field setting along the ZFS tensor axes. ENDOR spectra have been obtained for the first time also for the in-plane X- and Y-orientations of the ZFS tensor. This allowed a full determination of the hf-tensors of the three methine protons and one methyl group of 3P680. Based on the orientations of the axes of these hf-tensors, a unique orientation of the axes of the ZFS tensor of 3P680 in the Chl a molecular frame was obtained. These data serve as a structural basis for determining the orientation of 3P680 in the PS II protein complex by EPR on single crystals (see M. Kammel et al. in this issue). The data obtained represent the first complete set of the larger hf-tensors of the triplet state 3P680. They reflect the spin density distribution both in the highest occupied (HOMO) and lowest unoccupied (LUMO) orbitals. The data clearly confirm that 3P680 is a monomeric Chl a species at low temperature (T=10 K) used, as has been proposed earlier based on D- and E-values obtained from EPR and optically detected magnetic resonance (ODMR) studies. Comparison with the hf data for the cation and anion radicals of Chl a indicates a redistribution of spin densities in particular for the LUMO orbital of the triplet states. The electron spin distribution in the LUMO orbital is of special interest since it harbours the excited electron in the excited P680 singlet state, from which light-induced electron transfer proceeds. Observed shifts of hf couplings from individual nuclei of 3P680 as compared with 3Chl a in organic solution are of special interest, since they indicate specific protein interactions, e.g. hydrogen bonding, which might be used in future studies for assigning 3P680 to a particular chlorophyll molecule in PS II.