ScienceWeek

BIOLOGICAL CHEMISTRY: ON MOLYBDENUM IN ENZYMES

The following points are made by William N. Hunter (Nature 2004 430:736):

1) From bacteria to mammals, molybdenum (Mo) is crucial for survival. Enzymes that take advantage of the distinctive redox chemistry of this metal are involved in numerous metabolic reactions in the carbon, nitrogen and sulphur cycles(1,2). To be available for these reactions, however, water-soluble molybdate anions from the environment must be bound, transported into cells, and manipulated to place them in the correct context to exploit their chemical properties. This complex maneuvering of Mo involves an ancient and highly conserved biosynthetic pathway, and a key enzyme of the pathway in plants is called Cnx1. Recent work(3) reports two crystal structures of the Mo-binding domain of Cnx1. Unexpectedly, the structures suggest a new step in the pathway, involving an adenosine metabolite, and they implicate copper ions in the process as well -- two fortuitous and intriguing observations.

2) The vast majority of enzymes that make use of molybdenum (termed molybdoenzymes) do so through the Mo cofactor -- Moco for short. This cofactor contains a single Mo coordinated to an organic molecule called molybdopterin. The biosynthesis of Moco is highly conserved from bacteria through to mammals and involves four stages: first, conversion of a guanine nucleotide into a derivative termed precursor Z; second, conversion of precursor Z into molybdopterin; third, binding of Mo by molybdopterin, producing Moco; and fourth, attachment of a nucleotide moiety to Moco, forming molybdopterin guanine dinucleotide. In eukaryotes, Moco is considered the active form of the cofactor, but most bacterial enzymes require the molybdopterin guanine dinucleotide to make them functionally active.

3) How Moco is then inserted into molybdoenzymes is not yet understood because of the intrinsic instability of the chemical species concerned, but the intermediates and cofactor probably remain bound to other proteins throughout the biosynthetic process until the final incorporation of Moco to fully constitute the molybdoenzymes. The crystal structures of several proteins involved in Moco biosynthesis have been solved, and they mostly form oligomers(4). This, together with biochemical evidence showing that many of these proteins are involved in the formation of a heterogeneous complex, indicates that specific protein-protein interactions are crucial in the early stages of Moco biosynthesis(5).

4) The new crystal structures(3) are of Cnx1, which is homologous to gephyrin (in mammals) and MogA and MoeA (in bacteria). Once the molybdopterin moiety is formed at the end of the second stage, the metal ion has to be prepared and inserted into it --and this is where Cnx1 comes into play. Cnx1 contains two domains, termed G and E, that catalyze the transfer and insertion of Mo into the molybdopterin. Kuper et al(3) characterized domain G of Cnx1 structurally and biochemically under anaerobic conditions (because oxygen would cause some degradation).

References (abridged):

1. Stiefel, E. I. in Molybdenum Cofactors and Model Systems (eds Stiefel, E. I., Coucouvanis, D. & Newton, N. W.) 1-19 (Am. Chem. Soc., Washington DC, 1993)

2. Kisker, C., Schindelin, H. & Rees, D. C. Annu. Rev. Biochem. 66, 233-267 (1997)

3. Kuper, J. et al. Nature 430, 803-806 (2004)

4. Rizzi, M. & Schindelin, H. Curr. Opin. Struct. Biol. 12, 709-720 (2002)

5. Lake, M. W. et al. Nature 414, 325-329 (2001)

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BIOCHEMISTRY: ON METALLOPROTEINS

The following points are made by N.W. Aboelella et al (Science 2004 304:836):

1) Metalloproteins are involved in biological processes ranging from energy transduction to cellular signaling to modification of organic substrates. These processes often depend on the binding, activation, or generation of simple diatomic molecules like O2, N2, H2, NO, and CO at the active-site metal ion(s) (1). The metal-diatom adducts are fleeting intermediates in the catalytic pathways, making them difficult to characterize. However, knowing their chemical properties is key to understanding how the metalloenzymes function.

2) In generating nitric oxide (NO) from nitrite (NO2-) during anaerobic bacterial metabolism, the copper-containing nitrite reductase (NiR) plays a key role in the global nitrogen cycle. Previous studies of NiR in its resting state revealed three histidine ligands and a water molecule coordinated to the catalytic Cu(II) center (4,5). Nitrite binds at this center and is converted to NO, presumably via a copper-nitrosyl intermediate ([CuNO]2+), although this has not been observed. Indeed, structurally defined copper-nitrosyls are rare, the only monocopper examples being in amine oxidase and a synthetic complex LCuNO (L, tridentate anionic ligand), both of which feature end-on coordination of the NO ligand through the N atom.

3) Tocheva et al (2) have demonstrated that treatment of reduced NiR with NO in the absence of excess reductant yields a stable copper-nitrosyl ([CuNO]+) that features side-on coordination with the copper binding to both the N and O atoms. The observation of side-on binding is striking, because although it is established for synthetic O2 adducts of copper, such a binding mode for NO has only been seen in metastable complexes of other metals trapped at low temperatures. On the basis of electron paramagnetic resonance spectroscopy, the adduct is described as a Cu(II) complex of NO-, although more rigorous study is needed to confirm this assignment.

4) The side-on binding observed by Tocheva et al (2) in the NO adduct offers a possible solution to a mechanistic puzzle. Nitrite has been shown to bind to the oxidized NiR active-site Cu(II) via its O atoms, thus implicating an unprecedented O-bound copper-nitrosyl intermediate after reduction and dehydration. Tocheva et al (2) suggest instead that the specific arrangement of surrounding active-site residues provides hydrogen-bonding interactions that would favor a side-on bonded [CuNO]2+ catalytic intermediate similar to the structurally defined [CuNO]+ species. However, changing the redox state might perturb the binding interactions, and an alternative mechanism involving N-coordination of nitrite to the reduced enzyme and subsequent dehydration to a N-bound [CuNO]2+ species remains feasible. Distinguishing among these mechanisms is a challenge for future research.(3)

References (abridged):

1. J. A. McCleverty, T. J. Meyer, Eds., Comprehensive Coordination Chemistry II (Elsevier, Amsterdam, 2004), vol. 8

2. E. I. Tocheva, F. I. Rosell, A. G. Mauk, M. E. P. Murphy, Science 304, 867 (2004)

3. S. T. Prigge, B. A. Eipper, R. E. Mains, L. M. Amzel, Science 304, 864 (2004)

4. B. A. Averill, Chem. Rev. 96, 2951 (1996)

5. I. M. Wasser, S. de Vries, P. Moenne-Loccoz, I. Schroeder, K. D. Karlin, Chem. Rev. 102, 1201 (2002)

Science http://www.sciencemag.org

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ON NITROGENASE

The following points are made by Barry E. Smith (Science 2002 297:1654):

1) Given sufficient water, plant growth and therefore agricultural productivity is usually limited by the amount of bioavailable (fixed) nitrogen. Biological nitrogen fixation still contributes about half of the total nitrogen input to global agriculture, the rest principally coming from nitrogenous fertilizer produced chemically from the Haber-Bosch synthesis of ammonia. To produce the hydrogen gas together with the high temperatures and pressures needed for this chemical process, about 1% of the world's total annual energy supply has to be consumed. In marked contrast, a similar chemical process requiring only atmospheric temperature and pressure is carried out by nitrogen-fixing bacteria, many of which live in symbiotic association with legume plants. The secret of their success is the enzyme nitrogenase, which transforms atmospheric nitrogen gas (dinitrogen) into ammonia that plants can then use for growth. Many groups have tried for decades to determine how nitrogenase catalyzes this essential process.

2) Nitrogenase (2) consists of two essential metalloproteins: one, the iron (Fe) protein, is a very specific ATP-activated electron donor to the other, the molybdenum-iron (MoFe) protein. The MoFe protein contains two unique metallosulfur clusters: the P cluster [8Fe-7S] and the [Mo:7Fe:9S]:homocitrate iron-molybdenum (FeMo) cofactor cluster. About a decade ago, the first and relatively low-resolution (2.8 angstroms) crystal structure of the MoFe protein was reported (3). At this level of resolution, there were still some uncertainties about the structures of the metalloclusters. However, subsequent improvements in resolution to 2.0 angstroms (4) and then to 1.6 angstroms (5) yielded what seemed to be the accurate structure of the FeMo cofactor. The FeMo cofactor is bound to the MoFe protein through both a cysteine sulfur ligand (binding to the terminal tetrahedral iron atom) and a histidine ligand (binding to the molybdenum atom, which also binds to the homocitrate through its hydroxyl group and one carboxyl group). One of the features of the structure that has excited considerable interest is the trigonal nature of the other six iron atoms, which appear to be coordinately bound to only three atoms instead of the usual four or more atoms.

3) A high-resolution structure of part of bacterial nitrogenase reported by Einsle et al (1) yields some surprises about the biosynthesis and catalytic activity of this crucial metalloenzyme. Einsle et al. report the structure of the MoFe protein of bacterial nitrogenase at an improved resolution of 1.16 angstrom (1). This is a major achievement with a protein of this molecular size (~240,000 daltons).

References (abridged):

1. O. Einsle et al., Science 297, 1696 (2002).

2. B. E. Smith, in Advances in Inorganic Chemistry, A. G. Sykes, R. Cammack, Eds. (Academic Press, London, 1999), vol. 47, pp. 159-218.

3. J. S. Kim, D. C. Rees, Science 257, 1677 (1992).

4. J. W. Peters et al., Biochemistry 36, 1181 (1997).

5. S. M. Mayer, D. M. Lawson, C. A. Gormal, S. M. Roe, B. E. Smith, J. Mol. Biol. 292, 871 (1999).

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