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ScienceWeek
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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Related Material:
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).
Science http://www.sciencemag.org
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MARINE BIOLOGY: A BIOLOGICAL FUNCTION FOR CADMIUM
Notes by ScienceWeek
Marine microorganisms are largely responsible for the cycling and distribution of many nutrients in the sea, and it is the availability of these nutrients that in turn controls oceanic creation of organic matter (biomass) via photosynthesis or chemosynthesis (the creation process known as "primary production"). In addition to being true for major nutrients, this relationship is also true for essential trace elements. In oceanic systems, the vertical distribution (water column distribution) of many biologically important trace metals is thus similar to that of the major nutrients phosphate, nitrate, and silicate. At the ocean surface, where photosynthetic activity depletes algal nutrients, essential trace metals are present at extremely low concentrations, presumably as a result of biological uptake. Like those of major nutrients, these metal concentrations increase at depth because of decomposition of organic matter and remineralization.
The oceanic distribution of cadmium closely follows that of major nutrients such as phosphate, but the reasons for this "nutrient-like" distribution are unclear, since cadmium does not have an apparent biological function and is generally toxic to biological cells.
Diatoms, also called bacillariophytes, are microscopic unicellular eukaryotic algae differentiated into approximately 10,000 different species. (The term "eukaryotic" is applied to biological cells that have internal membrane-bound organelles such as a nucleus.)
Carbonic anhydrase (carbonate dehydratase) is an enzyme that catalyzes the reversible hydration of carbon dioxide to carbonic acid (or to bicarbonate ion at certain pH values). The enzyme is found in a wide range of living systems in various forms (isozymes). It is an intracellular enzyme with zinc as a cofactor.
The following points are made by T.W. Lane and F.M.M. Morel (Proc. Natl. Acad. Sci. 2000 97:4627):
1) The authors report evidence of a biological role for cadmium in the marine diatom Thalassiosira weissflogii under conditions of low zinc, a typical situation in the marine surface environment. The authors make the following points:
2) Addition of cadmium to zinc-limited cultures enhances the growth rate of T. weissflogii, particularly at low concentrations of carbon dioxide. This increase in growth rate is reflected in increased levels of cellular carbonic anhydrase activity, although the levels of TWCA1, the major intracellular zinc-requiring isoform of carbonic anhydrase in T. weissflogii remains low. Isotope cadmium label [(sup109)Cd] comigrates with a protein band that shows carbonic anhydrase activity and is distinct from native TWCA1. The levels of the cadmium protein are modulated by carbon dioxide in a manner consistent with a role for this enzyme in carbon acquisition. Purification of the carbonic anhydrase-active fraction leads to the isolation of a cadmium-containing protein of 43 kilodaltons. The authors suggest it is now clear that T. weissflogii expresses a cadmium-specific carbonic anhydrase, which, particularly under conditions of zinc limitation, can replace the zinc enzyme TWCA1 in its carbon-concentration mechanism.
3) The authors conclude: "It is very likely that substitution of metals in active metalloproteins and replacement of metalloenzymes by others containing other metals or none -- as observed for carbonic anhydrase -- are the mechanisms by which oceanic microorganisms have adapted to an environment of extremely low metal concentrations. It is thus not surprising that we should find common enzymes such as carbonic anhydrase with uncommon metal requirements. What we are observing is the unique biogeochemistry of the oceanic environment reflected in the unique biochemistry of its flora."
Proc. Nat. Acad. Sci. http://www.pnas.org
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