RAD50, an SMC family member with multiple roles in DNA break repair: how does ATP affect function?

The protein complex including Mre11, Rad50, and Nbs1 (MRN) functions in DNA double-strand break repair to recognize and process DNA ends as well as signal for cell cycle arrest. Amino acid sequence similarity and overall architecture make Rad50 a member of the structural maintenance of chromosome (SMC) protein family. Like SMC proteins, Rad50 function depends on ATP binding and hydrolysis. All current evidence indicates that ATP binding and hydrolysis cause architectural rearrangements in SMC protein complexes that are important for their functions in organizing DNA. In the case of the MRN complex, the functional significance of ATP binding and hydrolysis are not yet defined. Here we review the data on the ATP-dependent activities of MRN and their possible mechanistic significance. We present some speculation on the role of ATP for function of the MRN complex based on the similarities and differences in the molecular architecture of the Rad50-containing complexes and the SMC complexes condensin and cohesin.

RAD50 and NBS1 form a stable complex functional in DNA binding and tethering.

The RAD50/MRE11/NBS1 protein complex (RMN) plays an essential role during the early steps of DNA double-strand break (DSB) repair by homologous recombination. Previous data suggest that one important role for RMN in DSB repair is to provide a link between DNA ends. The striking architecture of the complex, a globular domain from which two extended coiled coils protrude, is essential for this function. Due to its DNA-binding activity, ability to form dimers and interact with both RAD50 and NBS1, MRE11 is considered to be crucial for formation and function of RMN. Here, we show the successful expression and purification of a stable complex containing only RAD50 and NBS1 (RN). The characteristic architecture of the complex was not affected by absence of MRE11. Although MRE11 is a DNA-binding protein it was not required for DNA binding per se or DNA-tethering activity of the complex. The stoichiometry of NBS1 in RMN and RN complexes was estimated by SFM-based volume analysis. These data show that in vitro, R, M and N form a variety of stable complexes with variable subunit composition and stoichiometry, which may be physiologically relevant in different aspects of RMN function.

An improved purification procedure for the soluble [NiFe]-hydrogenase of Ralstonia eutropha: new insights into its (in)stability and spectroscopic properties.

Infrared (IR) spectra in combination with chemical analyses have recently shown that the active Ni-Fe site of the soluble NAD(+)-reducing [NiFe]-hydrogenase from Ralstonia eutropha contains four cyanide groups and one carbon monoxide as ligands. Experiments presented here confirm this result, but show that a variable percentage of enzyme molecules loses one or two of the cyanide ligands from the active site during routine purification. For this reason the redox conditions during the purification have been optimized yielding hexameric enzyme preparations (HoxFUYHI(2)) with aerobic specific H(2)-NAD(+) activities of 150-185 mumol/min/mg of protein (up to 200% of the highest activity previously reported in the literature). The preparations were highly homogeneous in terms of the active site composition and showed superior IR spectra. IR spectro-electrochemical studies were consistent with the hypothesis that only reoxidation of the reduced enzyme with dioxygen leads to the inactive state, where it is believed that a peroxide group is bound to nickel. Electron paramagnetic resonance experiments showed that the radical signal from the NADH-reduced enzyme derives from the semiquinone form of the flavin (FMN-a) in the hydrogenase module (HoxYH dimer), but not of the flavin (FMN-b) in the NADH-dehydrogenase module (HoxFU dimer). It is further demonstrated that the hexameric enzyme remains active in the presence of NADPH and air, whereas NADH and air lead to rapid destruction of enzyme activity. It is proposed that the presence of NADPH in cells keeps the enzyme in the active state.

The soluble NAD+-Reducing [NiFe]-hydrogenase from Ralstonia eutropha H16 consists of six subunits and can be specifically activated by NADPH.

The soluble [NiFe]-hydrogenase (SH) of the facultative lithoautotrophic proteobacterium Ralstonia eutropha H16 has up to now been described as a heterotetrameric enzyme. The purified protein consists of two functionally distinct heterodimeric moieties. The HoxHY dimer represents the hydrogenase module, and the HoxFU dimer constitutes an NADH-dehydrogenase. In the bimodular form, the SH mediates reduction of NAD(+) at the expense of H(2). We have purified a new high-molecular-weight form of the SH which contains an additional subunit. This extra subunit was identified as the product of hoxI, a member of the SH gene cluster (hoxFUYHWI). Edman degradation, in combination with protein sequencing of the SH high-molecular-weight complex, established a subunit stoichiometry of HoxFUYHI(2). Cross-linking experiments indicated that the two HoxI subunits are the closest neighbors. The stability of the hexameric SH depended on the pH and the ionic strength of the buffer. The tetrameric form of the SH can be instantaneously activated with small amounts of NADH but not with NADPH. The hexameric form, however, was also activated by adding small amounts of NADPH. This suggests that HoxI provides a binding domain for NADPH. A specific reaction site for NADPH adds to the list of similarities between the SH and mitochondrial NADH:ubiquinone oxidoreductase (Complex I).

Structural and oxidation-state changes at its nonstandard Ni-Fe site during activation of the NAD-reducing hydrogenase from Ralstonia eutropha detected by X-ray absorption, EPR, and FTIR spectroscopy.

Structure and oxidation state of the Ni-Fe cofactor of the NAD-reducing soluble hydrogenase (SH) from Ralstonia eutropha were studied employing X-ray absorption spectroscopy (XAS) at the Ni K-edge, EPR, and FTIR spectroscopy. The SH comprises a nonstandard (CN)Ni-Fe(CN)(3)(CO) site; its hydrogen-cleavage reaction is resistant against inhibition by dioxygen and carbon monoxide. Simulations of the XANES and EXAFS regions of XAS spectra revealed that, in the oxidized SH, the Ni(II) is six-coordinated ((CN)O(3)S(2)); only two of the four conserved cysteines, which bind the Ni in standard Ni-Fe hydrogenases, provide thiol ligands to the Ni. Upon the exceptionally rapid reductive activation of the SH by NADH, an oxygen species is detached from the Ni; hydrogen may subsequently bind to the vacant coordination site. Prolonged reducing conditions cause the two thiols that are remote from the Ni in the native SH to become direct Ni ligands, creating a standardlike Ni(II)(CysS)(4) site, which could be further reduced to form the Ni-C (Ni(III)-H(-)) state. The Ni-C state does not seem to be involved in hydrogen cleavage. Two site-directed mutants (HoxH-I64A, HoxH-L118F) revealed structural changes at their Ni sites and were employed to further dissect the role of the extra CN ligand at the Ni. It is proposed that the predominant coordination by (CN),O ligands stabilizes the Ni(II) oxidation state throughout the catalytic cycle and is a prerequisite for the rapid activation of the SH in the presence of oxygen.

[NiFe]-hydrogenases of Ralstonia eutropha H16: modular enzymes for oxygen-tolerant biological hydrogen oxidation.

Recent research on hydrogenases has been notably motivated by a desire to utilize these remarkable hydrogen oxidation catalysts in biotechnological applications. Progress in the development of such applications is substantially hindered by the oxygen sensitivity of the majority of hydrogenases. This problem tends to inspire the study of organisms such as Ralstonia eutropha H16 that produce oxygen-tolerant [NiFe]-hydrogenases. R. eutropha H16 serves as an excellent model system in that it produces three distinct [NiFe]-hydrogenases that each serve unique physiological roles: a membrane-bound hydrogenase (MBH) coupled to the respiratory chain, a cytoplasmic, soluble hydrogenase (SH) able to generate reducing equivalents by reducing NAD+ at the expense of hydrogen, and a regulatory hydrogenase (RH) which acts in a signal transduction cascade to control hydrogenase gene transcription. This review will present recent results regarding the biosynthesis, regulation, structure, activity, and spectroscopy of these enzymes. This information will be discussed in light of the question how do organisms adapt the prototypical [NiFe]-hydrogenase system to function in the presence of oxygen.

The auxiliary protein HypX provides oxygen tolerance to the soluble [NiFe]-hydrogenase of ralstonia eutropha H16 by way of a cyanide ligand to nickel.

The hypX gene of the facultative lithoautotrophic bacterium Ralstonia eutropha is part of a cassette of accessory genes (the hyp cluster) required for the proper assembly of the active site of the [NiFe]-hydrogenases in the bacterium. A deletion of the hypX gene led to a severe growth retardation under lithoautotrophic conditions with 5 or 15% oxygen, when the growth was dependent on the activity of the soluble NAD+ -reducing hydrogenase. The enzymatic and infrared spectral properties of the soluble hydrogenase purified from a HypX-negative strain were compared with those from an enzyme purified from a HypX-positive strain. In activity assays under anaerobic conditions both enzyme preparations behaved the same. Under aerobic conditions, however, the mutant enzyme became irreversibly inactivated during H2 oxidation with NAD+ or benzyl viologen as the electron acceptor. Infrared spectra and chemical determination of cyanide showed that one of the four cyanide groups in the wild-type enzyme was missing in the mutant enzyme. The data are consistent with the proposal that the HypX protein is specifically involved in the biosynthetic pathway that delivers the nickel-bound cyanide. The data support the proposal that this cyanide is crucial for the enzyme to function under aerobic conditions.

The soluble [NiFe]-hydrogenase from Ralstonia eutropha contains four cyanides in its active site, one of which is responsible for the insensitivity towards oxygen.

Infrared spectra of (15)N-enriched preparations of the soluble cytoplasmic NAD(+)-reducing [NiFe]-hydrogenase from Ralstonia eutropha are presented. These spectra, together with chemical analyses, show that the Ni-Fe active site contains four cyanide groups and one carbon monoxide molecule. It is proposed that the active site is a (RS)(2)(CN)Ni(micro-RS)(2)Fe(CN)(3)(CO) centre (R=Cys) and that H(2) activation solely takes place on nickel. One of the two FMN groups (FMN-a) in the enzyme can be reversibly released upon reduction of the enzyme. It is now reported that at longer times also one of the cyanide groups, the one proposed to be bound to the nickel atom, could be removed from the enzyme. This process was irreversible and induced the inhibition of the enzyme activity by oxygen; the enzyme remained insensitive to carbon monoxide. The Ni-Fe active site was EPR undetectable under all conditions tested. It is concluded that the Ni-bound cyanide group is responsible for the oxygen insensitivity of the enzyme.

Selective release and function of one of the two FMN groups in the cytoplasmic NAD+-reducing [NiFe]-hydrogenase from Ralstonia eutropha.

The soluble, cytoplasmic NAD+-reducing [NiFe]-hydrogenase from Ralstonia eutropha is a heterotetrameric enzyme (HoxFUYH) and contains two FMN groups. The purified oxidized enzyme is inactive in the H2-NAD+ reaction, but can be activated by catalytic amounts of NADH. It was discovered that one of the FMN groups (FMN-a) is selectively released upon prolonged reduction of the enzyme with NADH. During this process, the enzyme maintained its tetrameric form, with one FMN group (FMN-b) firmly bound, but it lost its physiological activity--the reduction of NAD+ by H2. This activity could be reconstituted by the addition of excess FMN to the reduced enzyme. The rate of reduction of benzyl viologen by H2 was not dependent on the presence of FMN-a. Enzyme devoid of FMN-a could not be activated by NADH. As NADH-dehydrogenase activity was not dependent on the presence of FMN-a, and because FMN-b did not dissociate from the reduced enzyme, we conclude that FMN-b is functional in the NADH-dehydrogenase activity catalyzed by the HoxFU dimer. The possible function of FMN-a as a hydride acceptor in the hydrogenase reaction catalyzed by the HoxHY dimer is discussed.

Quantitative amino acid analysis of bovine NADH:ubiquinone oxidoreductase (Complex I) and related enzymes. Consequences for the number of prosthetic groups.

Bovine-heart NADH:ubiquinone oxidoreductase (EC 1.6.5.3; Complex I) is the first and most complicated enzyme in the mitochondrial respiratory chain. Biochemistry textbooks and virtually all literature on this enzyme state that it contains one FMN and at least four iron-sulfur clusters. We show here that this statement is incorrect as it is based on erroneous protein determinations. Quantitative amino acid analysis of the bovine Complex I, to our knowledge the first reported thus far, shows that the routine protein-determination methods used for the bovine Complex I overestimate its protein content by up to twofold. The FMN content of the preparations was determined to be at least 1.3-1.4 mol FMN/mol Complex I. The spin concentration of the electron paramagnetic resonance (EPR) signal ascribed to iron-sulfur cluster N2 was determined and accounted for 1.3-1.6 clusters per molecule of Complex I. These results experimentally confirm the hypothesis [FEBS Lett. 485 (2000) 1] that the bovine Complex I contains two FMN groups and two clusters N2. Also the protein content of preparations of the soluble NAD(+)-reducing [NiFe]-hydrogenase (EC 1.12.1.2) from Ralstonia eutropha, which shows clear evolutionary relationships with Complex I, scores too high by the colorimetric protein-determination methods. Determination of the FMN content and the spin concentration of the EPR signal of the [2Fe-2S] cluster shows that this hydrogenase also contains two FMN groups. A third enzyme (Ech), the membrane-bound [NiFe]-hydrogenase from Methanosarcina barkeri which shows an even stronger evolutionary relationship with Complex I, behaves rather normal in protein determinations and contains no detectable acid-extractable FMN in purified preparations.