Cartoon showing the movement of the iron sulfur protein of the bc1 complex, and other features of the Qo-site ballet
The Crofts Lab
Summary of Current Research Interests
Structure/function relationships in photosynthetic energy conversion; structure of membrane proteins; mechanism of energy conservation; photosynthesis in intact plants; energetic and informational fluxes of the biosphere, chronognosis, and evolutionary epistemology
Our work is based on the complementarity between biophysical, structural and molecular engineering approaches to the study of biological mechanism. Molecular engineering protocols have provided tools for modification of protein structure, but their utility is greatly increased by combining them with other approaches. Most importantly, knowledge of the structure of the target protein and the ability to assay the functional consequences of specific mutagenesis make it possible to explore the mechanism of catalysis at the molecular level.
- Ubiquinol:cytochrome c2 oxidoreductase (bc1 complex) of Rb. sphaeroides. As crystallographic structures become available, we are able to take advantage of spectroscopic techniques and protocols for protein engineering using PCR-cassette mutagenesis, that allow us to modify the three catalytic subunits, and answer specific questions about the structure, function and topology of the complex. These studies have revealed a novel mechanism of electron transfer in the bc1 complex, involving a dramatic domain movement of the head of the iron sulfur protein, and movement of quinone intermediates at the catalytic site; we are currently studying these using a variety of biophysical, biochemical and molecular engineering approaches, with local, national and international collaborative projects.
- In photosystem II, our present work centers on the function and structure of the two-electron gate through which the photosystem reduces the plastoquinone pool, and on the mechanism of oxygen evolution. We have developed structural models for the photosystem, biophysical assays for the reactions, and PCR-based techniques for analysis and sequence modification in the D1 subunit of photosystem II in Chlamydomonas.
- A more recent interest is in the role of informational fluxes in the biosphere. Attempts to incorporate these into the normal energetic fluxes has shown that one component, the semantic content, is not ammenable to standard thermodynamic description. This insubstantiality raises many interesting questions in relation to the mechanism of evolution, the nature of consciousness, and many aspects of philosophy centered on the "duality" question. Another aspect is 'information content' of physical systems, and how this plays out in discussions of "quantum reality"
Other interests include biophysical aspects of electron transfer and the coupling to ATP synthesis through the proton gradient, studies of the control of photosynthetic electron transfer in the coupled steady-state; the supramolecular organization of electron transfer chains; the mechanism and evolution of the cyt bc1/b6f family of complexes in plants and bacteria; the mechanism of action of inhibitors (including herbicides); and the role of thioredoxin in activation and control of chloroplast ATP-ase and other enzymes, and the control and modulation of photosynthesis in intact plants in the laboratory and under field conditions. We have developed novel instruments that allow us to explore individual reactions of the photosynthetic apparatus in intact plants. We are using these in the lab and in the field to try to understand how photosynthesis is regulated under natural conditions, including studies of the mechanisms of down regulation in strong light, photoinhibition, and response to environmental stress.
Structure of the bc1-complex.
- Berry, E.A., Zhang, Z., Huang, L.-S., Kuras, R., Guergova-Kuras, M. and Crofts, A.R. (1997) On the mechanism of ubiquinol oxidation by the bc1-complex: the role of the Rieske subunit, and its mobility.
Abstract, Meeting on "Reaction centers of photosynthetic purple bacteria: structure, spectroscopy, dynamics", Cadarache, June 1997. 3-D movie of the movement of the ISP presented at the meeting.
- Z. Zhang, L. Huang, V. M. Shulmeister, Y.-I. Chi, K.-K. Kim, L.-W. Hung, A. R. Crofts, E. A. Berry & S.-H. Kim. Electron transfer by domain movement in cytochrome bc1. Nature 392, 677 (1998).Abstract. Reprint (PDF)
- Crofts, A.R. and Berry, E.A. (1998) Structure and function of the cytochrome bc1 complex of mitochondria and photosynthetic bacteria. Curr. Opinions in Struc. Biol. 8, 501-509. Abstract, Full text, or reprint (PDF format).
- Crofts, A.R., Guergova-Kuras, M., Huang, L.-S., Kuras, R., Zhang, Z. and Berry, E.A.(1999) The mechanism of ubiquinol oxidation by the bc1 complex: the role of the iron sulfur protein, and its mobility. Biochemistry 38, 15791-15806. Reprint
- Izrailev, S., Crofts, A.R., Berry, E.A. and Schulten. K. (1999) Steered Molecular Dynamics simulation of the Rieske subunit motion in the cytochrome bc1 complex. Biophys. J. 77, 1753-1768. Reprint
- The bc1 complex in a Chime tutorial.
- Model structure for cytochrome b from prediction.
Mechanistic aspects of the Qo-site of the bc1-complex.
- Crofts, A.R., Barquera, B., Gennis, R.B., Kuras, R., Guergova-Kuras, M. and Berry, E.A. (1999) Mechanistic aspects of the Qo-site of the bc1 complex as revealed by mutagensis studies, and the crystallographic structure. In "The Phototrophic Prokaryotes", ed. by Peschek, G.A., Loeffelhardt, W., and Schmetterer, G. pp. 229-239. Plenum Publishing Corporation (New York - London - Washington, D.C. - Boston). (This paper was submitted at the IXth. International Symposium on Phototrophic Prokaryotes, Vienna,Sept. 1997. Publication was delayed until Jan 1999)
Abstract, Full text of paper, reprint (PDF format), or 3-Dmovie of the Qo-site mechanism suggested in this paper (635Kbyte)., or a voiced-over QuickTime movie of the mechnaism (5.3 Mbytes), kindly provided by John Allen. Alternatively, you can download compressed files for PC (zip, 2.1 Mbyte) or for the Mac (Stuffit, 2.05 Mbyte) by ftp.
- Crofts, A.R., Berry, E.A., Kuras, R., Guergova-Kuras, M., Hong, S. and Ugulava, N. (1998) Structures of the bc1 complex reveal dynamic aspects of mechanism. In "Photosynthesis: Mechanisms and Effects" (ed. by Garab, G.). Vol. III, pp. 1481-1486. Kluwer Academic Publ., Dordrecht/Boston/London. Pre-print (PDF format).
- Ugulava, N.B. and Crofts, A.R. (1998) CD-monitored redox titration of the Rieske Fe-S protein of Rhodobacter sphaeroides: pH dependence of the mid-point potential in isolated bc1 complex and in membranes. FEBS Lett.,440, 409-413. Reprint (PDF format)
- Kuras, R., Guergova-Kuras, M. and Crofts, A.R. (1998) Steps Toward Constructing a Cytochrome b6f Complex in the Purple Bacteria Rhodobacter sphaeroides: An Example of the Structural Plasticity of a Membrane Cytochrome. Biochemistry 37, 16280-16288. Reprint (PDF format)
- Crofts, A.R., Hong, S.J., Ugulava, N., Barquera, B., Gennis, R.B., Guergova-Kuras, M., and Berry, E. (1999) Pathways for proton release during ubihydroquinone oxidation by the bc1 complex. Proc. Natl. Acad. Sci. U.S.A., 96, 10021-10026. Reprint (PDF format)
- Guergova-Kuras, M., Salcedo-Hernandez, R., Bechmann, G., Kuras, R., Gennis, R.B. and Crofts, A.R. (1998) Expression and one-step purification of a fully active poly-histidine tagged bc1 complex from Rhodobacter sphaeroides. Protein Expression and Purification 15, 370-380. Reprint (PDF format)
- Crofts, A.R., Hong, S., Zhang, Z. and Berry, E.A. (1999) Physicochemical aspects of the movement of the Rieske iron sulfur protein during quinol oxidation by the bc1 complex. Biochemistry 38, 15827-15839. Reprint
- Crofts, A.R., Barquera, B., Gennis, R.B., Kuras, R., Guergova-Kuras, M. and Berry, E.A. (1999) The mechanism of ubiquinol oxidation by the bc1 complex: the different domains of the quinol binding pocket, and their role in mechanism, and the binding of inhibitors. Biochemistry 38, 15807-15826. Reprint
- Hong, S.J., Ugulava, N., Guergova-Kuras, M., and Crofts, A.R. (1999) The energy landscape for ubihydroquinone oxidation at the Qo-site of the bc1 complex in Rhodobacter sphaeroides. J. Biol. Chem. 274: 33931-33944. Reprint, or Download an interactive program to explore Marcus curves for Qo electron transfer reactions
- Guergova-Kuras, M., Kuras, R., Ugulava, N., Hadad, I., and Crofts, A.R. (2000) Specific mutagenesis of the Rieske iron sulfur protein in Rhodobacter sphaeroides shows that both thermodynamic gradient and the pK of the oxidized form determine the rate of quinol oxidation by the bc1 complex. Biochemistry 39, 7436-7444 Reprint
- Berry, E., Guergova-Kuras, M., Huang, L.-S. and Crofts, A.R. (2000) Structure and Function of Cytochrome bc complexes. Annu. Rev. Biochem. 69, 1007-77. Reprint
- Crofts, A.R., Guergova-Kuras, M., Kuras, R., Ugulava, N., Li, J. and Hong, S. (2000) Proton-coupled electron transfer at the Qo-site: what type of mechanism can account for the high activation barrier? Biochim. Biophys. Acta 1459, 456-466. Reprint
- Shinkarev, V.P., Ugulava, N.B., Takahashi, E., Crofts, A.R. and Wraight, C.A. (2000) Aspartate-187 of cytochrome b is not needed for DCCD inhibition of ubiquinol: cytochrome c oxidoreductase in Rhodobacter sphaeroides chromatophores. Biochemistry 39, 14232-14237.. Reprint
- Shinkarev, V.P., Ugulava, N.B., Crofts, A.R. and Wraight, C.A. (2000) DCCD inhibits the reactions of the iron-sulfur protein in Rhodobacter sphaeroides chromatophores. Biochemistry, 39, 16206-16212. Reprint
- Vladimir P. Shinkarev, Antony R. Crofts, and Colin A. Wraight (2001) The Electric Field Generated by Photosynthetic Reaction Center Induces Rapid Reversed Electron Transfer in the bc1 Complex, Biochemistry 40, 12584-12590. Reprint
- Crofts, A.R., Guergova-Kuras, M., Ugulava, N., Kuras, R. and Hong, S. (2001) Proton processing at the Qo-site of the bc1 complex of Rhodobacter sphaeroides. Proc XIIth. Int. Cong. Photosynt., Brisbane, Aug 2001. In press. Reprint
- Samoilova, R.I., Kolling, D., Uzawa, T., Iwasaki, T., Crofts, A.R. and Dikanov, S.A. (2001) The interaction of the Rieske iron sulfur protein with occupants of the Qo-site of the bc1 complex, probed by 1D and 2D Electron Spin Echo Envelope Modulation. J. Biol. Chem., 277, 4605-4608. Reprint
- Muller, F., Crofts, A.R. and Kramer, D.M. (2002) Multiple Q-cycle bypass reactions at the Qo-site of the cytochrome bc1 complex. Biochemistry 41, 7866-7874. Reprint
- Shinkarev, V.P., Kolling, D.R.J., Miller, T.J. and Crofts, A.R. (2002) Modulation of the Midpoint Potential of the [2Fe-2S] Rieske Iron Sulfur Center by Qo Occupants in the bc1 Complex. Biochemistry 41, 14372-14382. Reprint
- Crofts, A.R., Shinkarev, V.P., Dikanov, S.A., Samoilova, R.I., Kolling, D. (2002) Interactions of quinone with the iron-sulfur protein of the bc1 complex: Is the mechanism spring-loaded? Biochim. Biophys. Acta, 1555, 48-53. Reprint
- Crofts, A.R., Shinkarev, V.P., Kolling, D.R.J. and Hong, S. (2003) The modified Q-cycle explains the apparent mismatch between the kinetics of reduction of cytochromes c1 and bH in the bc1 complex. J. Biol. Chem. 278, 36191-36201. Reprint
- Kolling, D.R.J., Samoilova, R.I., Holland, J.T., Berry, E.A, Dikanov, S.A., and Crofts, A.R. (2003) Exploration of ligands to the Qi-site semiquinone in the bc1 complex using high resolution EPR. J. Biol. Chem. 278, 39747-39754. Reprint
- Zu, Y., Manon, M.-J., Couture, M.M.-J., Kolling, D.R.J., Crofts, A.R., Eltis, L.D., Fee, J.A. and Hirst, J. (2003) The reduction potentials of Rieske clusters: the importance of the coupling between oxidation state and histidine protonation state. Biochemistry 42, 12400-12408. Reprint
- Wang, Q., Crofts, A.R. and Padua, G.W. (2003) Protein-Lipid Interactions in Zein Films Investigated by Surface Plasmon Resonance. J. Agric. Food Chem. 51, 7439-7444
- Crofts, A.R. (2004) Proton-coupled electron transfer at the Qo-site of the bc1 complex controls the rate of ubihydroquinone oxidation. Biochim. Biophys. Acta 1655, 77-92. Reprint
- Dikanov, S.A., Samoilova, R.I., Kolling, D.R.J., Holland, J.T. and Crofts, A.R. (2004) Hydrogen bonds involved in binding the Qi-site semiquinone in the bc1 complex, identified through deuterium exchange using pulsed EPR. J. Biol. Chem. 279, 15814-15823. Reprint
- Iwasaki, T., Kounosu, A., Kolling, D.R.J., Crofts, A.R., Dikanov, S.A., Jin, A., Imai, T. and Urushiyama, A. (2004) Characterization of the pH-Dependent Resonance Raman Transitions of Archaeal and Bacterial Rieske [2Fe-2S] Proteins. J. Am. Chem. Soc. 126, 4788-4789. Reprint
- Shinkarev, V.P., Crofts, A.R. and Wraight, C.A. (2005) Spectral Analysis of the bc1 Complex Components in Situ: Beyond the Traditional Difference Approach. Biochim. Biophys. Acta, 1757, 67-77.Reprint
- Tang, J.Y., Zielinski, R.E., Zangerl, A.R., Crofts, A.R., Berenbaum, M.R. and DeLucia, E.H. (2006) The differential effects of herbivory by first and fourth instars of Trichoplusia ni (Lepidoptera : Noctuidae) on photosynthesis in Arabidopsis thaliana. J. Exp. Botany. 57, 527-536. Reprint
- Crofts, A.R., Lhee, S., Crofts, S.B., Cheng, J. and Rose, S. (2006) Proton pumping in the bc1 complex: A new gating mechanism that prevents short circuits. Biochim. Biophys. Acta, 1757, 1019-1034. Reprint
- Iwasaki, T., Kounosu, A., Kolling, D.R.J., Lhee, S., Crofts, A.R., Dikanov, S.A., Uchiyama, T., Kumasaka, T., Ishikawa, H., Kono, M, Imai, T. and Urushiyama, A. (2006) Resonance Raman characterization of archaeal and bacterial Rieske protein variants with modified hydrogen bond network around the [2Fe-2S] center. Protein Science 15, 2019–2024. Reprint
- Shinkarev, V.P., Crofts, A.R. and Wraight, C.A. (2006) Spectral and kinetic resolution of the bc1 complex components in situ: A simple and robust alternative to the traditional difference wavelength approach. Biochim. Biophys. Acta 1757, 273-283. Reprint
- Shinkarev, V.P., Crofts, A.R. and Wraight, C.A. (2006) In situ kinetics of cytochromes c1 and c2. Biochemistry 45, 7897-7903. Reprint
- Dikanov, S.A., Kolling, D.R.J., Endeward, B., Samoilova, R.I., Prisner, T.F. Satish K. Nair, S.K. and Crofts, A.R. (2006) Identification of Hydrogen Bonds to the Rieske Cluster through the Weakly Coupled Nitrogens Detected by Electron Spin Echo Envelope Modulation Spectroscopy. J. Biol. Chem. 281, 27416-27425. Reprint
- Kolling, D.R.J, Brunzelle, J.S., Lhee, S., Crofts, A.R. and Nair, S.K. (2007) Atomic resolution structures of Rieske iron-sulfur protein: role of hydrogen bonds in tuning redox potential of iron-sulfur clusters. Structure 15, 29-38. Reprint
- Dikanov, S.A., Holland, J.T., Endeward, B., Kolling, D.R.J., Samoilova, R.I., Prisner, T.F. and Crofts, A.R. (2007) Hydrogen bonds between nitrogen donors and the semiquinone in the Qi-site of the bc1 complex. J. Biol. Chem. 282, 25831–25841. Reprint
- Crofts, A.R. and Rose, S. (2007) Marcus treatment of endergonic reactions: A commentary. Biochim. Biophys. Acta 1767, 1228–1232. Reprint
- Crofts, A.R. (2007) Life, information, entropy, and time. Complexity, 13, 14-50. Reprint
The modified Q-cycle.
Outline of modified Q-cycle.
Working models of the modified Q-cycle, including movies and Chime-based animation.
3-D Movie showing the mechanism we have suggested for the Qo-site.
Relation between structure and function explored using molecular engineering. (Paper from the Xth. Internatl. Photosynthesis Congress, Montpelier, with amplified Figs.)
Marcus-Bronsted program for analysis of energy-gap law parameters in Qo-site reaction. Files available include executable program, Visual Basic source code, and Manual.
Heterogeneity in local domains determines reaction characteristics for bimolecular processes - a response to the supercomplex hypothesis.
- Crofts, A. R., Guergova-Kuras, M. and Hong, S. (1998) Chromatophore heterogeneity explains effects previously attributed to supercomplexes. Photosynth. Res. 55, 357-362. Reprint (pdf)
- Chromatophore heterogeneity explains observations on the electron transfer kinetics on illumination of Rhodobacter sphaeroides previously attributed to supercomplexes, - a more extended discussion of the above topic, including additional data.
- Two notes arising from a correspondence in Trends in Microbiology on supercomplexes:
- Crofts, A.R. (2000) Photosynthesis in Rhodobacter sphaeroides. Trends in Microbiol. 8, 105. Reprint
- Crofts, A.R. (2000) Response from Crofts. Trends in Microbiol. 8, 107-108. Reprint
PSAAM package, and Walsh-Crofts Structural Propensity Indices
Current interests in photosystem II.
- Minagawa, J. and Crofts, A.R. (1994) A robust protocol for site-directed mutagenesis of the D1 protein of Chlamydomonas reinhardtii: a PCR-spliced psbA gene in a plasmid conferring spectinomycin resistance was introduced into a psbA deletion strain. Photosynth. Research, 42, 121-132.
- Minagawa, J., Kramer, D.M., Kanazawa, A. and Crofts, A.R. (1996) Donor-side photoinhibition in photosystem II from Chlamydomonas reinhardtii upon mutation of tyrosine-Z in the D1 polypeptide to phenylalaine. FEBS Lett. 389, 199-202.
- Jin Xiong, J., Minagawa, J., Crofts, A.R. and Govindjee (1998) Loss of Inhibition by Formate in Newly Constructed Photosystem II D1 Mutants, D1-R257E and D1-R257M, of Chlamydomonas reinhardtii. Biochim. Biophys. Acta, 1365, 473-491.
- Mechanism of Non-photochemical Fluorescence Quenching
- Hypothesis paper: A Molecular Mechanism for QE-quenching
- Kramer, D.M. and Crofts A.R. (1996) Control and Measurement of Photosynthetic Electron Transport in Vivo. In Advances in Photosynthesis, Vol. 5: Photosynthesis and the Environment (Baker, N. R., ed.). pp. 25-66. Kluwer Academic Publishers, Dordrecht/Boston/London.
Energetics of the biosphere, chronognosis, and evolutionary epistemology
Movie showing herbicide uptake by a leaf (MPEG)Click here for more recent fluoresence movies from Jim Fenton, Brian Wade and Lloyd Wax.Download Multimedia Xplorer for MPEG (and most other image and video formats) viewing.
Protein ViewersChime Protein Viewer for Netscape. Click HELP link for download insructions.
Protein Data Viewer for Windows (PDVWIN Package)E-mail to A.R. Crofts
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TeachingBiological Energy Conversion Biophysics 354
This course is taught in the Fall semester by ARC, and is available on the WWW. The course contains many Chime tutorials on proteins of interest in bioenergetics and photosysnthesis.
Other useful stuff.
Chime and Rasmol LinksRasmol is a program for viewing macromolecular structures coded in the Brookhaven Protein Data Bank (PDB) format. Chime is a plugin for Netscape (versions 3.0 and higher) which uses many of the Rasmol commands to allow viewing of PDB files (and many other formats) in the browser.All PDB files used in Chime can be downloaded using the File option under the Chime menu. These can then be explored in greater detail using Rasmol.