oleg

Montana State University
Department of Chemistry and Biochemistry
230 Chemistry and Biochemistry & Building
Bozeman, MT 59717

Email: olegz@chemistry.montana.edu

Research Interests

I came here in Prof. John Peters’ lab at Montana State University from Russia in 2005 to study hydrogenases from phototrophic bacteria. Here I become very involved in structural and biochemical characterization of hydrogenases from phototrophic bacteria and their role in the detoxification of metal ions in bacterial cells. I am also involved in the characterization of hydrogenases encapsulated in silica gel materials and development of photo hydrogen producing catalysts, photosensitizers and sensor systems based on hydrogenases and nitrogenase. Recently I have been working on structural characterization of the nitrogenase MoFe – protein to understand the catalytic mechanism of this enzyme, as well as bifurcating transhydrogenases NfnI and NfnII from thermophiles.

Bozeman, Montana is a very great place for people who like hiking, fishing and skiing. I enjoy hiking on mountains in summer and skiing in winter.


 

Structural characterization of hydrogenases from phototrophic bacteria

Figure1

We have been working on the structural characterization of hydrogenase from the purple sulfur bacteria Thiocapsa roseopersicina.  This hydrogenase is a heterodimer consist of large and small subunits with molecular weights of 64 and 34 kD. The hydrogenase from T. roseopersicinais highly stable during storage under aerobic conditions with a half-life at 4° C of more than two months. It is highly thermal stable as well and remains active up to temperatures of 80°C.  The hydrogenase from T. roseopersicina with high activity and stability is promising as a hydrogen-activating catalyst for development of fuel cells and hydrogen sensors based on direct bioelectrocatalysis.

Transmission electron microscopy data of the hydrogenase from T. roseopersicina indicated that it is organized in complicated super molecular complexes consisting of 6 αβ-dimer units. Together with Professor L. Tang from the University of Kansas, performing cryoreconstruction experiments, we found that the super molecular complex consists of 2 agglomerated rings composed of three heterodimers (6 heterodimers total) (Figure 1).

Currently, collaborating with the Pushchino group (Russia) we have been working on purification as well as spectroscopic, and structural characterization of a hox encoded [NiFe]-hydrogenase from Chloroflexus aurantiacus.

Metal detoxification and nano particles formation by microorganisms and their hydrogenases

Figure2

A common microbial strategy for detoxifying metals involves redox transformation which often results in metal precipitation and/or immobilization. The focus of our research is the influence of ionic nickel [Ni(II)] on growth of the purple sulfur bacterium,Thiocapsa roseopersicina.  The results show that Ni(II) in the bulk medium at micromolar concentrations results in growth inhibition; specifically, an increase in the lag phase of growth, a decrease in the specific growth rate, and a decrease in total protein concentration when compared to growth controls containing no added Ni(II).  The inhibitory effects of Ni(II) on the growth of T. roseopersicina could be partially overcome by the addition of hydrogen (H2) gas.  However, the inhibitory effects of Ni(II) on the growth of T. roseopersicina were not alleviated by H2 in a strain containing deletions in all hydrogenase-encoding genes.  Transmission electron micrographs of wild-type T. roseopersicinagrown in the presence of Ni(II) and H2 revealed a significantly greater number of dense nanoparticulates associated with the cells when compared to wild-type cells grown in the absence of H2and hydrogenase-mutant strains grown in the presence of H2.  X-ray diffraction and vibrating sample magnetometry of the dense nanoparticles indicated the presence of zero-valent Ni, suggesting Ni(II) reduction (Figure 2).

Figure3

Purified T. roseopersicina hyn-encoded hydrogenase catalyzed the formation of zero valent Ni particles in vitro, suggesting a role for this hydrogenase in Ni(II) reduction in vivo (Figure 3).  Collectively, these results suggest a link between H2metabolism, Ni(II) tolerance, and Ni(II) reduction in T. roseopersicina.

Encapsulation of hydrogenases in sol-gel material doped with carbon nanotubes

scheme1

Much attention has been directed towards developing hydrogen fuel technology in order to realize the tremendous potential hydrogen gas has as a clean, renewable, and reliable fuel source.  The enzyme hydrogenase can catalyze the production of hydrogen from protons and electrons. We have demonstrated that silica-derived gels can be used to stabilize hydrogenases.  We have been investigating the conditions for encapsulation to promote significantly enhanced levels of activities by doping gels with multi-walled carbon nanotubes, polyethylene glycol and methyl viologen (Scheme 1).  Optimization of these dopants results in hydrogen production levels comparable to those observed for the enzyme in solution.  Encapsulation of active hydrogenases in solid materials is a promising advancement towards their use in hydrogen producing catalytic materials applications.

Development of light harvesting hydrogen producing catalyst stable to oxygen based on hydrogenases and photosensitizer

Figure4

The exposed surfaces of the protein-based H2 producing catalyst systems (both the hydrogenase and synthetic protein cage systems) provide a rich template for covalent attachment of light harvesting antennae systems including either molecular, colloidal, or solid surfaces. The overall goal of this research is to maximize the light harvesting capacity of each self-assembled protein-based catalyst for optimized and directed electron transfer to the protein catalyst.  For the hydrogenases, covalent attachment is not simple since the ability to heterologously express the enzymes of interest in a manner that allows the purification of large amounts of homogenous preparations of enzyme is not straightforward.  We have therefore been using a two pronged approach involving pilot work targeted at providing a preliminary analysis of the effects of coupling model light harvesting complexes to both [NiFe]- and [FeFe]-hydrogenases that are the targets of our studies and in parallel improving the means to express hydrogenase enzymes and engineer modification sites in an informed manner.
   Our intitial studies on covalent attachment has been using the [NiFe]-hydrogenase from Thiocapsa roseopersicina as a model system because it is highly stable in comparison with other [NiFe]-hydrogenases.  In our pilot work, the enhanced stability of this enzyme allows it to retain a signficantly larger percentage of activity that other [NiFe]-hydrogenase or [FeFe]-hydrogenases upon labeling.  The photocatalytic ruthenium complex ([Ru(bpy)2(5-NH2phen)]2+) (Ru(II)-complex) has been covalently attached to stable [NiFe]- hydrogenase from T. roseopersicina using 1-ethyl-3-(3-dimethylaminopropyl carbodiimide (EDC) which links amino groups of the complex to exposed carboxyl groups of the hydrogenase.  We have optimized the conditions for the light depended hydrogen production by the covalently modified protein with the Ru(II) complex (Figure 4). We have been able to show that labeled T. roseopersicina hydrogenase retains ~50% of its activity after labeling and highest activities are observed at pH 5.0 under a nitrogen atmosphere and irradiated by 150,000 Lux light.  So far we were unable to demonstrate light dependent reaction by labeled hydrogenase without an electron carrier; however we have found that attachment of the Ru(II) complex to the hydrogenase greatly enhances light dependent hydrogenase activity (22 times) over that of the equivalent Ru(II) complex concentrations in solution.

Education

1998-2004

  • Ph.D. in Biochemistry, Institute of Basic Biological Problems of the Russian Academy of Sciences, Pushchino, Moscow Region, Russian Federation.
  • Ph.D. thesis: "Redox interaction of hydrogenase from phototrophic bacteria with metals" (2004)

1996-1998

  • M.Sc. in Biochemistry, Pushchino State University, Pushchino, Moscow Region, Russian Federation.
  • M.Sc. thesis: "Effect of various light conditions on the PAL (phenilalanine ammonia lyase) activity in pea and arabidopsis leafs." (1998)
  • 1991-1996

  • M.Sc. in Plant physiology, Leo Tolstoy's Tula State Teachers Training University, Tula,
  • M.Sc.: "Influence of chromium salts and UV-light on the habitus of wheat and leguminous plants." (1996)

Research Experience

2005-present

Research Scientist, Postdoctoral Researcher
Department of Chemistry and Biochemistry, Montana State University, Bozeman, MT.

Prof. John Peters' lab

Laboratory of Biochemistry and Biotechnology of Phototrophic Microorganisms of the Institute of Basic Biological Problems of the Russian Academy of Sciences, Pushchino, Moscow Region, Russian Federation.

Dr. Nikolay Zorin's Lab

  • Study the structure and crystallization hydrogenase from phototrophic bacteria, purification hydrogenases from different bacteria.
  • Structural characterization of MoFe protein of nitrogenise with amino acid substitutions in the active site to understand a catalytic mechanism of nitrogenise.
  • Study properties and stability of hydrogenases encapsulated in silica gel.
  • Investigation the transformation of metal ions by hydrogenases and bacteria.

1998-2005

Research Fellow
Laboratory of Biochemistry and Biotechnology of Phototrophic Microorganisms of the Institute of Basic Biological Problems of the Russian Academy of Sciences, Pushchino, Moscow Region, Russian Federation.

Prof. Ivan Gogotov’s Lab

  • hydrogen metabolism in photosynthetic bacteria and cyanobacteria;
  • reduction and oxidation metals by oxidoreductase;
  • investigation of enzyme properties;
  • hydrogen enzyme electrode

1996-1998

Junior Researcher
Laboratory of regulation of plant growth and photosynthesis of the Institute of Basic Biological Problems of the Russian Academy of Sciences, Pushchino, Moscow Region, Russian Federation.

Dr. Evgeny Muzafarov's Lab

  • Purification and properties of PAL (phenilalanine ammonia lyase); effect of light condition on PAL activity.

Current Collaborations

  • Timothy E. Elgren, Hamilton College, USA
  • Trevor Douglas, Montana State University, USA
  • Liang Tang, University of Kansas, USA
  • Nikolay A Zorin, IBBP RAS, RF

Selected Publications

  1. Schut GJ, Zadvornyy O, Wu CH, Peters JW, Boyd ES, Adams MW. The role of geochemistry and energetics in the evolution of modern respiratory complexes from a proton-reducing ancestor. Biochim Biophys Acta. 2016 Jul;1857(7):958-70. doi: 10.1016/j.bbabio.2016.01.010. Epub 2016 Jan 22. Review. PubMed PMID: 26808919.
  2. Artz JH, White SN, Zadvornyy OA, Fugate CJ, Hicks D, Gauss GH, Posewitz MC, Boyd ES, Peters JW. Biochemical and Structural Properties of a Thermostable Mercuric Ion Reductase from Metallosphaera sedula. Front Bioeng Biotechnol. 2015  Jul 13;3:97. doi: 10.3389/fbioe.2015.00097. eCollection 2015. PubMed PMID: 26217660; PubMed Central PMCID: PMC4500099.
  3. Zadvornyy OA, Boyd ES, Posewitz MC, Zorin NA, Peters JW. Biochemical and Structural Characterization of Enolase from Chloroflexus aurantiacus: Evidence for a Thermophilic Origin. Front Bioeng Biotechnol. 2015 Jun 1;3:74. doi: 10.3389/fbioe.2015.00074. eCollection 2015. PubMed PMID: 26082925; PubMed Central PMCID: PMC4450660.
  4. Danyal K, Rasmussen AJ, Keable SM, Inglet BS, Shaw S, Zadvornyy OA, Duval S, Dean DR, Raugei S, Peters JW, Seefeldt LC. Fe protein-independent substrate reduction by nitrogenase MoFe protein variants. Biochemistry. 2015 Apr 21;54(15):2456-62. doi: 10.1021/acs.biochem.5b00140. Epub 2015 Apr 7. PubMed PMID: 25831270.
  5. Swanson KD, Ratzloff MW, Mulder DW, Artz JH, Ghose S, Hoffman A, White S, Zadvornyy OA, Broderick JB, Bothner B, King PW, Peters JW. [FeFe]-hydrogenase oxygen inactivation is initiated at the H cluster 2Fe subcluster. J Am Chem Soc. 2015 Feb 11;137(5):1809-16. doi: 10.1021/ja510169s. Epub 2015 Jan 29. PubMed PMID: 25579778
  6. Therien JB, Zadvornyy OA, Posewitz MC, Bryant DA, Peters JW. Growth of Chlamydomonas reinhardtii in acetate-free medium when co-cultured with alginate-encapsulated, acetate-producing strains of Synechococcus sp. PCC 7002. Biotechnol Biofuels. 2014 Oct 18;7(1):154. doi: 10.1186/s13068-014-0154-2. eCollection 2014. PubMed PMID: 25364380; PubMed Central PMCID: PMC4216383. 
  7. Cohen AE, Soltis SM, González A, Aguila L, Alonso-Mori R, Barnes CO, Baxter EL, Brehmer W, Brewster AS, Brunger AT, Calero G, Chang JF, Chollet M, Ehrensberger P, Eriksson TL, Feng Y, Hattne J, Hedman B, Hollenbeck M, Holton JM, Keable S, Kobilka BK, Kovaleva EG, Kruse AC, Lemke HT, Lin G, Lyubimov AY, Manglik A, Mathews II, McPhillips SE, Nelson S, Peters JW, Sauter NK, Smith CA, Song J, Stevenson HP, Tsai Y, Uervirojnangkoorn M, Vinetsky V, Wakatsuki S, Weis WI, Zadvornyy OA, Zeldin OB, Zhu D, Hodgson KO. Goniometer-based femtosecond crystallography with X-ray free electron lasers. Proc Natl Acad Sci U S A. 2014 Dec 2;111(48):17122-7. doi: 10.1073/pnas.1418733111. Epub 2014 Oct 31. PubMed PMID: 25362050; PubMed Central PMCID: PMC4260607.
  8. Oleg A. Zadvornyy, Janice E. Lucon, Robin Gerlach, Nikolay A. Zorin, Trevor Douglas, Timothy E. Elgren , John W. Peters. (2012) Photo-induced H2 production by [NiFe]-hydrogenase from T. roseopersicina covalently linked to a Ru(II) photosensitizer. J. Inorg. Biochem. 106(1), 151-155.(http://www.sciencedirect.com/science/article/pii/S0162013411002686)
  9. Gogotov I.N., Zadvornyy O.A., Zorin N.A., Serebriakova L.T. Bacterial hydrogenases. In Phototrophic microorganisms. Edit Galchenko B.F. Moscow:Max-Press, 2010, v. 15, pp 260-289 (Russian).
  10. Oleg A. Zadvorny, Amy M. Barrows, Nikolay A. Zorin, John W. Peters and Timothy E. Elgren. (2010) High level of hydrogen production activity achieved for hydrogenase encapsulated in sol–gel material doped with carbon nanotubes. J. Mater. Chem., 20, 1065–1067. (http://www.rsc.org/Publishing/Journals/JM/article.asp?doi=b922296k).
  11. Oleg A. Zadvornyy, Mark Allen, Susan K. Brumfield, Zack Varpness, Eric S. Boyd, Nikolay A. Zorin, Larisa Serebriakova, Trevor Douglas, and John W. Peters. (2010) Hydrogen enhances nickel tolerance in the purple sulfur bacterium Thiocapsa roseopersicina. Environ. Scien. Thechnol., 44, 834-840. (http://pubs.acs.org/doi/abs/10.1021/es901580n).
  12. Zadvorny O.A., Zorin N.A., Gogotov I.N. (2006) Transformation of metals and metal ions by hydrogenases from phototrophic bacteria. Archives of Microbiology, Jan; 184 (5), 279-285. (http://www.ncbi.nlm.nih.gov/pubmed/16283252).
  13. Elgren T.E., Zadvorny O.A., Brecht E., Douglas T., Zorin N.A., Maroney M.J., Peters J.W. (2005) Immobilization of active hydrogenases by encapsulation in polymeric porous gels. Nano letters, 5 (10), 2085. (http://www.ncbi.nlm.nih.gov/pubmed/16218742).
  14. Zadvorny O.A., Zorin N.A., Gogotov I.N. and Gorlenko V.M. (2004) Properties of stable hydrogenase from the purple sulfur bacterium Lamprobacter modestohalophilus. Biochemistry (Mosc). Feb; 69 (2), 164-9. (http://www.ncbi.nlm.nih.gov/pubmed/15000682).
  15. Morozov S.V., Karyakina E.E., Zadvorny O.A., Zorin N.A., Varfolomeev S.D., Karyakin A.A. (2002) Bioelectrocatalysis by T. roseopersicina hydrogenase immobilised on different carbon supports. Russian Journal of Electrochemistry, 38 (1), 97-102.
  16. Zadvorny O.A., Zorin N.A., Gogotov I.N. (2000) The effect of metal ions on hydrogenase of purple sulphur bacterium Thiocapsa roseopersicina. Biochemistry-Moscow. 65 (11), 1287-1291. (http://www.ncbi.nlm.nih.gov/pubmed/11112845).
  17. Gogotov I.N., Zorin N.A., Zadvorny O.A. (1999) Accumulation and extraction of metals by phototrophic microorganisms. In Environment and Soils: selective lectures of VIII-IX Russian Schools., Moscow: Politex, pp. 238-251 (Russian)
  18. Zorin N.A., Gogotov I.N., Zadvorny O.A. (2002) Redox reaction of bacterial hydrogenase with metals and metal ions. Microelements in Medicine, 3 (2), 66-68. (Russian)