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Abstracts Special Lectures
Journal of Inorganic Biochemistry 86 (2001)
17
Fluorescent probes for studying the neurochemistry of nitric oxide and zinc Stephen J. Lippard, Shawn C. Burdette, Katherine J. Franz, Scott R. Hilderbrand Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139. USA, (e-mail: lippard@lippard, m it. edu) Elucidation of the bioinorganic chemistry occurring in the central nervous system represents a major challenge in understanding neurological functions such as reflexes, learning, and memory formation. Transition metals and radical species are implicated in many neurological disorders including Alzheimer's disease (AD) and Parkinson's disease. The neurochemistry of Na ÷, K ÷, and Ca 2÷ has been extensively investigated; however, other inorganic species such as zinc (Zn 2+) and nitric oxide (NO) have been examined to a much lesser degree. In order to facilitate the study of intracellular Zn 2÷, we prepared and characterized Zinpyr-1 and Zinpyr-2, two new fluorescein-based sensors, in collaboration with the laboratory of R. Y. Tsien at UCSD. These new sensors exhibit a fluorescence increase (3-5 fold at pH 7) upon Zn 2+ binding, have optical properties amenable to intracellular work, and membrane permeability permitting them to be loaded passively into cells without further functionalization. The sensors were particularly useful for imaging the fluorescence response of zinc-filled vesicles that exist in the CA-3 hippocampal cells in rat brain. This work, carried out in collaboration with C. A. Frederickson, demonstrates that a Zinpyr-l-treated live rat hippocampal brain slice exhibits clear evidence for the zinc-rich glutamatergic vesicles, which appear as bright spots in the image. The staining by the dye shows what has not been previously visible, namely, the intravesicular zinc in situ in the neurons. Our initial investigations into NO sensors have revealed a new paradigm for small molecule sensing based on the quenching properties of transition metals with partially filled d orbitals. The fluorescence of the [Co(DATI-4)] complex, where DATI is a derivative of dansyl aminotroponiminate, is quenched by the partially filled d orbital of the cobalt center. Upon reaction of the complex with NO, a tetrahedral dinitrosyl species is formed, where one of the DATI arms dissociates from the metal. The interruption of the communication between the dansyl-containing ligand fragment and cobalt results in a restoration of fluorescence. This work was supported by grants from the NSF, the McKnight Foundation, and MIT.
A multitude of normal and abnormal heme peroxidases in plants Karen G. Welinder, Laurent Duroux Institute of Biotechnology, Aalborg University, Sohngaardsholmsvej 49, DK-9000 Denmark (e-mail: welinder@bio, auc. dk) Genome, transcriptome, proteome and metabolome are the key words in a new era of large-scale and systematic research in biology. High-throughput technologies have provided access to a wealth of novel gene sequences. The complete genome sequence of the model plant Arabidopsis has revealed close to 26,000 genes (Nature (Dec 2000) 408, 796-815). For almost 10 years many of the corresponding eDNA-clones (expressed sequence tag, EST) have been available for recombinant expression and structure-function studies. We have found 50 cDNA clones encoding different class III peroxidases and recently identified a total of 73 prx genes in the Arabidopsis genome. An amazing struc~ral diversity was found, with 50% of the putative mature peroxidases showing less than 38% amino acid, and only 5% showing greater than 50% identity. Several plant peroxidases have been produced in E. coli and their crystal structures, spectroscopic and kinetic properties analyzed. (Supported by the EU BIO4 and TMR programs, and the Danish SNF and SJVF Research Councils.)
18
Journal of Inorganic Biochemistry 86 (2001)
The use of heavy element isotope fractionation in the study of the uptake of elements into cells R.J.P. W i U i a m s g, K. O n i o n s a, X. Z h u a, B.K. B u r g e s s b, G. W. Canters c, E. de W a a l c, A. M a t t h e w s d, B. Salvato e, U. W e s e r f,
aEarth Sciences Dept., University of Oxford, UK; bUniversity of California, Irvine, USA; CUniversity of Leiden, THE NETHERLANDS; aHebrew University, ISRAEL; eUniversity of Padua, ITAL Y; f University of Tubingen, GERMANY gInorganic Chemistry Dept., University of Oxford, South Parks Road, Oxford OX1 3QR UK In order to follow the pathways of uptake of heavy metal elements (or of heavy non-metals) into cells t we have been testing the use of isotope fractionation. The procedure is just the same as that which has been used extensively for following H, C, N, O etc. fractionation except that new mass spectrometric methods have been employed. The elements studied so far have been mainly Fe and Cu. As is general in isotope fractionation studies there are very different degrees of separation due to equilibrium and kinetically controlled paths. We have therefore looked first at model fractionations in equilibria in some minerals where notable separation was seen, and in chromatographic separations (see Glueckauf2), where very small fractionations were found per theoretical plate. Fractionation in kinetically controlled redox changes between iron chloride and dipyrridyl complexes have also been studied as models for similar processes in cellular incorporation. Here large fractionation was found. In cells we have looked at the uptake of (1) iron into ferredoxin Av Fd. 1 of azobacter vinlandii and into hemoglobins of higher animals, and of (2) copper into azurin ofpseudomonas aeruginosa and into yeast metallothionein and its superoxide dismutase and into octopus hemocyanin. As these processes are multi-step we have also examined the uptake of copper into the same isolated azurin, now initially prepared in genetically modified E. Coll. All the experiments with biological processes showed considerable fractionation. The overall impression is that isotope studies will allow us to follow heavy element uptake into cells but as complex ion species are not readily defined exactly due to hydration and fast exchange amongst similar species it will be difficult to develop a full theory such as is used in say H/D fractionations in organic chemistry. Even so it should be possible to follow the pathway of incorporation. Frafisto da Silva, J.J.R. and Williams, R.J.P., The Biological Chemistry of the Elements 2"d edition Oxford University Press, Oxford (2001); 2. Glueckauf, E., Trans. Faraday Soc., 54, 1203-1205 (1958)
Sunrise Lecture Bioinoganic chemistry and the biogeochemical cycles Edward I. Stiefel Corporate Strategic Research, ExxonMobil Research and Engineering, 1545 Rt 22E, Annandale, NJ 08801, USA Life on Earth depends upon and, to a significant extent, influences the biogeochemical cycling of the major elements: C, H, O, N, S, and P. The cycles for the first five of these elements involve redox reactions and biological systems have played major roles in the evolution of these cycles and in the maintenance of the levels of carbon, hydrogen, nitrogen, oxygen and sulfur compounds in the atmosphere and the oceans. Key steps in the various cycles are catalyzed by metalloenzymes that contain Fe, Mn, Co, Ni, Cu, Zn, and Mo. Under certain conditions, Cd and W play significant roles. This lecture presents an overview of the biogeochemical cycles on Earth emphasizing: the co-evolution of the cycles and life; the interactions between the cycles; the key role that transition elements play in the major cycles as components of critical redox-active sites in metalloenzymes; and the perturbations to the natural cycles that are occurring due to human activities. Environmental bioinorganic chemistry has a critical role to play in the elucidation of the past history, current state, and ultimate fate of the biogeochemical cycles.
17
Fluorescent probes for studying the neurochemistry of nitric oxide and zinc Stephen J. Lippard, Shawn C. Burdette, Katherine J. Franz, Scott R. Hilderbrand Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139. USA, (e-mail: lippard@lippard, m it. edu) Elucidation of the bioinorganic chemistry occurring in the central nervous system represents a major challenge in understanding neurological functions such as reflexes, learning, and memory formation. Transition metals and radical species are implicated in many neurological disorders including Alzheimer's disease (AD) and Parkinson's disease. The neurochemistry of Na ÷, K ÷, and Ca 2÷ has been extensively investigated; however, other inorganic species such as zinc (Zn 2+) and nitric oxide (NO) have been examined to a much lesser degree. In order to facilitate the study of intracellular Zn 2÷, we prepared and characterized Zinpyr-1 and Zinpyr-2, two new fluorescein-based sensors, in collaboration with the laboratory of R. Y. Tsien at UCSD. These new sensors exhibit a fluorescence increase (3-5 fold at pH 7) upon Zn 2+ binding, have optical properties amenable to intracellular work, and membrane permeability permitting them to be loaded passively into cells without further functionalization. The sensors were particularly useful for imaging the fluorescence response of zinc-filled vesicles that exist in the CA-3 hippocampal cells in rat brain. This work, carried out in collaboration with C. A. Frederickson, demonstrates that a Zinpyr-l-treated live rat hippocampal brain slice exhibits clear evidence for the zinc-rich glutamatergic vesicles, which appear as bright spots in the image. The staining by the dye shows what has not been previously visible, namely, the intravesicular zinc in situ in the neurons. Our initial investigations into NO sensors have revealed a new paradigm for small molecule sensing based on the quenching properties of transition metals with partially filled d orbitals. The fluorescence of the [Co(DATI-4)] complex, where DATI is a derivative of dansyl aminotroponiminate, is quenched by the partially filled d orbital of the cobalt center. Upon reaction of the complex with NO, a tetrahedral dinitrosyl species is formed, where one of the DATI arms dissociates from the metal. The interruption of the communication between the dansyl-containing ligand fragment and cobalt results in a restoration of fluorescence. This work was supported by grants from the NSF, the McKnight Foundation, and MIT.
A multitude of normal and abnormal heme peroxidases in plants Karen G. Welinder, Laurent Duroux Institute of Biotechnology, Aalborg University, Sohngaardsholmsvej 49, DK-9000 Denmark (e-mail: welinder@bio, auc. dk) Genome, transcriptome, proteome and metabolome are the key words in a new era of large-scale and systematic research in biology. High-throughput technologies have provided access to a wealth of novel gene sequences. The complete genome sequence of the model plant Arabidopsis has revealed close to 26,000 genes (Nature (Dec 2000) 408, 796-815). For almost 10 years many of the corresponding eDNA-clones (expressed sequence tag, EST) have been available for recombinant expression and structure-function studies. We have found 50 cDNA clones encoding different class III peroxidases and recently identified a total of 73 prx genes in the Arabidopsis genome. An amazing struc~ral diversity was found, with 50% of the putative mature peroxidases showing less than 38% amino acid, and only 5% showing greater than 50% identity. Several plant peroxidases have been produced in E. coli and their crystal structures, spectroscopic and kinetic properties analyzed. (Supported by the EU BIO4 and TMR programs, and the Danish SNF and SJVF Research Councils.)
18
Journal of Inorganic Biochemistry 86 (2001)
The use of heavy element isotope fractionation in the study of the uptake of elements into cells R.J.P. W i U i a m s g, K. O n i o n s a, X. Z h u a, B.K. B u r g e s s b, G. W. Canters c, E. de W a a l c, A. M a t t h e w s d, B. Salvato e, U. W e s e r f,
aEarth Sciences Dept., University of Oxford, UK; bUniversity of California, Irvine, USA; CUniversity of Leiden, THE NETHERLANDS; aHebrew University, ISRAEL; eUniversity of Padua, ITAL Y; f University of Tubingen, GERMANY gInorganic Chemistry Dept., University of Oxford, South Parks Road, Oxford OX1 3QR UK In order to follow the pathways of uptake of heavy metal elements (or of heavy non-metals) into cells t we have been testing the use of isotope fractionation. The procedure is just the same as that which has been used extensively for following H, C, N, O etc. fractionation except that new mass spectrometric methods have been employed. The elements studied so far have been mainly Fe and Cu. As is general in isotope fractionation studies there are very different degrees of separation due to equilibrium and kinetically controlled paths. We have therefore looked first at model fractionations in equilibria in some minerals where notable separation was seen, and in chromatographic separations (see Glueckauf2), where very small fractionations were found per theoretical plate. Fractionation in kinetically controlled redox changes between iron chloride and dipyrridyl complexes have also been studied as models for similar processes in cellular incorporation. Here large fractionation was found. In cells we have looked at the uptake of (1) iron into ferredoxin Av Fd. 1 of azobacter vinlandii and into hemoglobins of higher animals, and of (2) copper into azurin ofpseudomonas aeruginosa and into yeast metallothionein and its superoxide dismutase and into octopus hemocyanin. As these processes are multi-step we have also examined the uptake of copper into the same isolated azurin, now initially prepared in genetically modified E. Coll. All the experiments with biological processes showed considerable fractionation. The overall impression is that isotope studies will allow us to follow heavy element uptake into cells but as complex ion species are not readily defined exactly due to hydration and fast exchange amongst similar species it will be difficult to develop a full theory such as is used in say H/D fractionations in organic chemistry. Even so it should be possible to follow the pathway of incorporation. Frafisto da Silva, J.J.R. and Williams, R.J.P., The Biological Chemistry of the Elements 2"d edition Oxford University Press, Oxford (2001); 2. Glueckauf, E., Trans. Faraday Soc., 54, 1203-1205 (1958)
Sunrise Lecture Bioinoganic chemistry and the biogeochemical cycles Edward I. Stiefel Corporate Strategic Research, ExxonMobil Research and Engineering, 1545 Rt 22E, Annandale, NJ 08801, USA Life on Earth depends upon and, to a significant extent, influences the biogeochemical cycling of the major elements: C, H, O, N, S, and P. The cycles for the first five of these elements involve redox reactions and biological systems have played major roles in the evolution of these cycles and in the maintenance of the levels of carbon, hydrogen, nitrogen, oxygen and sulfur compounds in the atmosphere and the oceans. Key steps in the various cycles are catalyzed by metalloenzymes that contain Fe, Mn, Co, Ni, Cu, Zn, and Mo. Under certain conditions, Cd and W play significant roles. This lecture presents an overview of the biogeochemical cycles on Earth emphasizing: the co-evolution of the cycles and life; the interactions between the cycles; the key role that transition elements play in the major cycles as components of critical redox-active sites in metalloenzymes; and the perturbations to the natural cycles that are occurring due to human activities. Environmental bioinorganic chemistry has a critical role to play in the elucidation of the past history, current state, and ultimate fate of the biogeochemical cycles.