The N-terminus of the human RecQL4 helicase is a homeodomain-like DNA interaction motif

The RecQL4 helicase is involved in the maintenance of genome integrity and DNA replication. Mutations in the human RecQL4 gene cause the Rothmund–Thomson, RAPADILINO and Baller–Gerold syndromes. Mouse models and experiments in human and Xenopus have proven the N-terminal part of RecQL4 to be vital for cell growth. We have identified the first 54 amino acids of RecQL4 (RecQL4_N54) as the minimum interaction region with human TopBP1. The solution structure of RecQL4_N54 was determined by heteronuclear liquid–state nuclear magnetic resonance (NMR) spectroscopy (PDB 2KMU; backbone root-mean-square deviation 0.73 Å). Despite low-sequence homology, the well-defined structure carries an overall helical fold similar to homeodomain DNA-binding proteins but lacks their archetypical, minor groove-binding N-terminal extension. Sequence comparison indicates that this N-terminal homeodomain-like fold is a common hallmark of metazoan RecQL4 and yeast Sld2 DNA replication initiation factors. RecQL4_N54 binds DNA without noticeable sequence specificity yet with apparent preference for branched over double-stranded (ds) or single-stranded (ss) DNA. NMR chemical shift perturbation observed upon titration with Y-shaped, ssDNA and dsDNA shows a major contribution of helix α3 to DNA binding, and additional arginine side chain interactions for the ss and Y-shaped DNA.     

Spare quinones in the QB cavity of crystallized photosystem II from Thermosynechococcus elongatus

The recent crystallographic structure at 3.0 Å resolution of PSII from Thermosynechococcus elongatus has revealed a cavity in the protein which connects the membrane phase to the binding pocket of the secondary plastoquinone Q_B. The cavity may serve as a quinone diffusion pathway. By fluorescence methods, electron transfer at the donor and acceptor sides was investigated in the same membrane-free PSII core particle preparation from T. elongatus prior to and after crystallization; PSII membrane fragments from spinach were studied as a reference. The data suggest selective enrichment of those PSII centers in the crystal that are intact with respect to O₂ evolution at the manganese-calcium complex of water oxidation and with respect to the integrity of the quinone binding site. One and more functional quinone molecules (per PSII monomer) besides of Q_A and Q_B were found in the crystallized PSII. We propose that the extra quinones are located in the Q_B cavity and serve as a PSII intrinsic pool of electron acceptors.     

Eight steps preceding O-O bond formation in oxygenic photosynthesis--a basic reaction cycle of the Photosystem II manganese complex

In oxygenic photosynthesis, water is split at a Mn(4)Ca complex bound to the proteins of photosystem II (PSII). Powered by four quanta of visible light, four electrons and four protons are removed from two water molecules before dioxygen is released. By this process, water becomes an inexhaustible source of the protons and electrons needed for primary biomass formation. On the basis of structural and spectroscopic data, we recently have introduced a basic reaction cycle of water oxidation which extends the classical S-state cycle [B. Kok, B. Forbush, M. McGloin, Cooperation of charges in photosynthetic O2 evolution- I. A linear four-step mechanism, Photochem. Photobiol. 11 (1970) 457-475] by taking into account also the role and sequence of deprotonation events [H. Dau, M. Haumann, Reaction cycle of photosynthetic water oxidation in plants and cyanobacteria, Science 312 (2006) 1471-1472]. We propose that the outwardly convoluted and irregular events of the classical S-state cycle are governed by a simple underlying principle: protons and electrons are removed strictly alternately from the Mn complex. Starting in I(0), eight successive steps of alternate proton and electron removal lead to I(8) and only then the O-O bond is formed. Thus not only four oxidizing equivalents, but also four bases are accumulated prior to the onset of dioxygen formation. After reviewing the kinetic properties of the individual S-state transition, we show that the proposed basic model explains a large body of experimental results straightforwardly. Furthermore we discuss how the I-cycle model addresses the redox-potential problem of PSII water oxidation and we propose that the accumulated bases facilitate dioxygen formation by acting as proton acceptors.     

Eight steps preceding O-O bond formation in oxygenic photosynthesis—A basic reaction cycle of the Photosystem II manganese complex

In oxygenic photosynthesis, water is split at a Mn₄Ca complex bound to the proteins of photosystem II (PSII). Powered by four quanta of visible light, four electrons and four protons are removed from two water molecules before dioxygen is released. By this process, water becomes an inexhaustible source of the protons and electrons needed for primary biomass formation. On the basis of structural and spectroscopic data, we recently have introduced a basic reaction cycle of water oxidation which extends the classical S-state cycle [B. Kok, B. Forbush, M. McGloin, Cooperation of charges in photosynthetic O2 evolution- I. A linear four-step mechanism, Photochem. Photobiol. 11 (1970) 457-475] by taking into account also the role and sequence of deprotonation events [H. Dau, M. Haumann, Reaction cycle of photosynthetic water oxidation in plants and cyanobacteria, Science 312 (2006) 1471-1472]. We propose that the outwardly convoluted and irregular events of the classical S-state cycle are governed by a simple underlying principle: protons and electrons are removed strictly alternately from the Mn complex. Starting in I₀, eight successive steps of alternate proton and electron removal lead to I₈ and only then the O-O bond is formed. Thus not only four oxidizing equivalents, but also four bases are accumulated prior to the onset of dioxygen formation. After reviewing the kinetic properties of the individual S-state transition, we show that the proposed basic model explains a large body of experimental results straightforwardly. Furthermore we discuss how the I-cycle model addresses the redox-potential problem of PSII water oxidation and we propose that the accumulated bases facilitate dioxygen formation by acting as proton acceptors.     

High-valent [MnFe] and [FeFe] cofactors in ribonucleotide reductases

Ribonucleotide reductases (RNRs) are essential for DNA synthesis in most organisms. In class-Ic RNR from Chlamydia trachomatis (Ct), a MnFe cofactor in subunit R2 forms the site required for enzyme activity, instead of an FeFe cofactor plus a redox-active tyrosine in class-Ia RNRs, for example in mouse (Mus musculus, Mm). For R2 proteins from Ct and Mm, either grown in the presence of, or reconstituted with Mn and Fe ions, structural and electronic properties of higher valence MnFe and FeFe sites were determined by X-ray absorption spectroscopy and complementary techniques, in combination with bond-valence-sum and density functional theory calculations. At least ten different cofactor species could be tentatively distinguished. In Ct R2, two different Mn(IV)Fe(III) site configurations were assigned either L(4)Mn(IV)(μO)(2)Fe(III)L(4) (metal-metal distance of ~2.75?, L = ligand) prevailing in metal-grown R2, or L(4)Mn(IV)(μO)(μOH)Fe(III)L(4) (~2.90?) dominating in metal-reconstituted R2. Specific spectroscopic features were attributed to an Fe(IV)Fe(III) site (~2.55?) with a L(4)Fe(IV)(μO)(2)Fe(III)L(3) core structure. Several Mn,Fe(III)Fe(III) (~2.9-3.1?) and Mn,Fe(III)Fe(II) species (~3.3-3.4?) likely showed 5-coordinated Mn(III) or Fe(III). Rapid X-ray photoreduction of iron and shorter metal-metal distances in the high-valent states suggested radiation-induced modifications in most crystal structures of R2. The actual configuration of the MnFe and FeFe cofactors seems to depend on assembly sequences, bound metal type, valence state, and previous catalytic activity involving subunit R1. In Ct R2, the protonation of a bridging oxide in the Mn(IV)(μO)(μOH)Fe(III) core may be important for preventing premature site reduction and initiation of the radical chemistry in R1.     

Enthalpy changes during photosynthetic water oxidation tracked by time-resolved calorimetry using a photothermal beam deflection technique

The energetics of the individual reaction steps in the catalytic cycle of photosynthetic water oxidation at the Mn₄Ca complex of photosystem II (PSII) are of prime interest. We studied the electron transfer reactions in oxygen-evolving PSII membrane particles from spinach by a photothermal beam deflection technique, allowing for time-resolved calorimetry in the micro- to millisecond domain. For an ideal quantum yield of 100%, the enthalpy change, ΔH, coupled to the formation of the radical pair (where Y_Z is Tyr-161 of the D1 subunit of PSII) is estimated as −820 ± 250 meV. For a lower quantum yield of 70%, the enthalpy change is estimated to be −400 ± 250 meV. The observed nonthermal signal possibly is due to a contraction of the PSII protein volume (apparent ΔV of about −13 Å³). For the first time, the enthalpy change of the O₂-evolving transition of the S-state cycle was monitored directly. Surprisingly, the reaction is only slightly exergonic. A value of ΔH(S₃ ⇒ S₀) of −210 meV is estimated, but also an enthalpy change of zero is within the error range. A prominent nonthermal photothermal beam deflection signal (apparent ΔV of about +42 Å³) may reflect O₂ and proton release from the manganese complex, but also reorganization of the protein matrix.     

Evidence for impaired hydrogen-bonding of tyrosine YZ in calcium-depleted photosystem II

Photosystem II (PS II) evolves oxygen from two bound water molecules in a four-stepped reaction that is driven by four quanta of light, each oxidizing the chlorophyll moiety P680 to yield P+680. When starting from its dark equilibrium (mainly state S1), the catalytic center can be clocked through its redox states (S0ellipsisS4) by a series of short flashes of light. The center involves at least a Mn4-cluster and a special tyrosine residue, named YZ, as redox cofactors plus two essential ionic cofactors, Cl- and Ca2+. Centers which have lost Ca2+ do not evolve oxygen. We investigated the stepped progression in dark-adapted PS II core particles after the removal of Ca2+. YZ was oxidized from the first flash on. The difference spectrum of YZ-->YoxZ differed from the one in competent centers, where it has been ascribed to a hydrogen-bonded tyrosinate. The rate of the electron transfer from YZ to P+680 was slowed down by three orders of magnitude and its kinetic isotope effect rose up from 1.1 to 2.5. Proton release into the bulk was now a prerequisite for the electron transfer from YZ to P+680. On the basis of these results and similar effects in Mn-(plus Ca2+-)depleted PS II (M. Haumann et al., Biochemistry, 38 (1999) 1258-1267) we conclude that the presence of Ca2+ in the catalytic center is required to tune the apparent pK of a base cluster, B, to which YZ is linked by hydrogen bonds. The deposition of a proton on B within close proximity of YZ (not its release into the bulk!) is a necessary condition for the reduction in nanoseconds of P+680 and for the functioning of water oxidation. The removal of Ca2+ rises the pK of B, thereby disturbing the hydrogen bonded structure of YZB.     

Electrostatics and proton transfer in photosynthetic water oxidation

Photosystem II (PSII) oxidizes two water molecules to yield dioxygen plus four protons. Dioxygen is released during the last out of four sequential oxidation steps of the catalytic centre (S(0) --> S(1), S(1) --> S(2), S(2) --> S(3), S(3) --> S(4) --> S(0)). The release of the chemically produced protons is blurred by transient, highly variable and electrostatically triggered proton transfer at the periphery (Bohr effect). The extent of the latter transiently amounts to more than one H(+)/e(-) under certain conditions and this is understood in terms of electrostatics. By kinetic analyses of electron-proton transfer and electrochromism, we discriminated between Bohr-effect and chemically produced protons and arrived at a distribution of the latter over the oxidation steps of 1 : 0 : 1 : 2. During the oxidation of tyr-161 on subunit D1 (Y(Z)), its phenolic proton is not normally released into the bulk. Instead, it is shared with and confined in a hydrogen-bonded cluster. This notion is difficult to reconcile with proposed mechanisms where Y(Z) acts as a hydrogen acceptor for bound water. Only in manganese (Mn) depleted PSII is the proton released into the bulk and this changes the rate of electron transfer between Y(Z) and the primary donor of PSII P(+)(680) from electron to proton controlled. D1-His190, the proposed centre of the hydrogen-bonded cluster around Y(Z), is probably further remote from Y(Z) than previously thought, because substitution of D1-Glu189, its direct neighbour, by Gln, Arg or Lys is without effect on the electron transfer from Y(Z) to P(+)(680) (in nanoseconds) and from the Mn cluster to Y(ox)(Z).     

NiFe] and [FeS] cofactors in the membrane-bound hydrogenase of Ralstonia eutropha investigated by X-ray absorption spectroscopy: insights into O(2)-tolerant H(2) cleavage

Molecular features that allow certain [NiFe] hydrogenases to catalyze the conversion of molecular hydrogen (H(2)) in the presence of dioxygen (O(2)) were investigated. Using X-ray absorption spectroscopy (XAS), we compared the [NiFe] active site and FeS clusters in the O(2)-tolerant membrane-bound hydrogenase (MBH) of Ralstonia eutropha and the O(2)-sensitive periplasmic hydrogenase (PH) of Desulfovibrio gigas. Fe-XAS indicated an unusual complement of iron-sulfur centers in the MBH, likely based on a specific structure of the FeS cluster proximal to the active site. This cluster is a [4Fe4S] cubane in PH. For MBH, it comprises less than ~2.7 ? Fe-Fe distances and additional longer vectors of ≥3.4 ?, consistent with an Fe trimer with a more isolated Fe ion. Ni-XAS indicated a similar architecture of the [NiFe] site in MBH and PH, featuring Ni coordination by four thiolates of conserved cysteines, i.e., in the fully reduced state (Ni-SR). For oxidized states, short Ni-μO bonds due to Ni-Fe bridging oxygen species were detected in the Ni-B state of the MBH and in the Ni-A state of the PH. Furthermore, a bridging sulfenate (CysSO) is suggested for an inactive state (Ni(ia)-S) of the MBH. We propose that the O(2) tolerance of the MBH is mainly based on a dedicated electron donation from a modified proximal FeS cluster to the active site, which may favor formation of the rapidly reactivated Ni-B state instead of the slowly reactivated Ni-A state. Thereby, the catalytic activity of the MBH is facilitated in the presence of both H(2) and O(2).     

The structure of the Ni-Fe site in the isolated HoxC subunit of the hydrogen-sensing hydrogenase from Ralstonia eutropha

The regulatory Ni-Fe hydrogenase (RH) from Ralstonia eutropha which forms a [HoxBC]2 complex functions as a hydrogen sensor under aerobic conditions. We have studied a novel Strep-tag isolate of the RH large subunit, HoxC(ST), which lacks the Fe-S clusters of HoxB, allowing for structure determination of the catalytic site by X-ray absorption spectroscopy both at the Ni and, for the first time, also at the Fe K-edge. This technique, together with Fourier-transform infrared spectroscopy, revealed a Ni-Fe site with [O1(CysS)2Ni(II)(mu-SCys)2Fe(II)(CN)2(CO)] structure in about 50% of HoxC(ST) and a [(CysS)2Fe(II)(CN)2(CO)] site lacking Ni in the remainder protein. Possibly both sites may be intermediates in the maturation process of the RH.