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.

The bc₁ complex of Rhodobacter sphaeroides

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 Q_o-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 Q_o-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 Q_o-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 Q_o-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 Q_o 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 Q_o-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 Q_o-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 Q_o-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 Q_o-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 Q_o 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 Q_i-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.

• A speculative model of the Q-cycle mechanism for the bc₁ complex, animated in Chime (This version has extensive notes). This model also shows the mechanism for release of protons from the Q_o-site.
• The model in an annotated, animated gif. This version does not require Chime, but the annotations are necessarily brief.
• Animate gif movie showing the coulombic gating model suggested to minimize bypass reactions at the Q_o-site.

 Working models of the modified Q-cycle, including movies and Chime-based animation.

 3-D Movie showing the mechanism we have suggested for the Q_o-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 Q_o-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

Structural Prediction.

Function and Structure of 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.

How Plants Protect themselves against High Light.

• Mechanism of Non-photochemical Fluorescence Quenching
• Hypothesis paper: A Molecular Mechanism for Q_E-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

• An essay on the information content of the biosphere, and some interesting consequences of attempts to quantify this.
• Disentangling entanglement: a discussion of the epistemological underpinnings of the "entanglement" problem in physics.(A more extended version of this paper has been submitted for publication, which includes a program simulating entanglement experiments that demonstrates a 'local realistic' representation of a correlated pair of photons leading to the expectations of Bell's quantum mechanical model (and violation of the expectations of his 'local realistic' model) so long as the law of Malus is correctly implemented.)
• PowerPoint presentation on "Semantic Transmission and the emergent Mind", from a PITP Workshop om "Emergence".

Protein Viewers

Protein Data Viewer for Windows (PDVWIN Package)

A.R. Crofts' Home Page

Back to Welcome page

Teaching

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.

• Protein Data Bank
• Annotated Guideto the Brookhaven Protein Data Bank.
• NCBI GenBank with Entrez.
• European Molecular Biology Laboratory(EMBL).
• Web Resourcesfor Protein Sciences, by the Stephen White Laboratory, UC Irvine
• Primer on Molecular Genetics (Department of Energy)
• Department of Biochemistry
• Biophysics Home Page
• Microbiology Home Page
• The bc₁-complex Home Page
• Life Sciencs at UIUC.
• Molecular and Cellular Biology, UIUC
• Sunsite.
• NIHGMS
• NSF
• DOE
• USDA
• Photosynthesis Research Center, ASU
 
• Download PDVWIN
• Download PSAAM

• Download Rasmolfrom the Rasmol Homepage.
• Download Chime
• How to use Chime, a brief tutorial.
• Draft Chime Manual.
• Draft Rasmol Manual (version 2.6).