The School of Molecular and Cellular Biology at the University of Illinois at Urbana-Champaign

We use biophysical, spectroscopic, computational, structural and molecular engineering approaches to the study of the mechanism of the bc₁ complex. The utility of molecular engineering for modification of protein structure is greatly increased by combining this with other approaches. Most importantly, knowledge of the structure of the target protein from crystallography and spectroscopy and the ability to assay the functional consequences of specific mutagenesis make it possible to explore the mechanism of catalysis at the molecular level. The bc₁ complex is central to all major energy conversion pathways. It oxidizes ubiquinol, reduces cytochrome c, and uses the work potential to form the proton gradient that drives ATP synthesis. We study the enzyme from the photosynthetic bacterium Rhodobacter sphaeroides. In membrane vesicles, we can measure kinetics in situ by photoactivation of the photosynthetic reaction center, which generates the substrates for the complex. With crystallographic structures now available, we take advantage of spectroscopic techniques and protocols for protein engineering that allow us to modify the three catalytic subunits, and ask specific questions about the structure using kinetic spectrophotometry, and rapid-mix freeze-quench approaches and EPR spectroscopy to trap and measure reaction intermediates, to probe function and topology of the complex, and the detailed mechanism of partial processes. Our studies have revealed novel mechanisms of electron transfer in the bc₁ complex; one involves a dramatic domain movement of the extrinsic domain of the iron sulfur protein; more tentative is another proposal for movement of a semiquinone intermediate at the catalytic site. Detailed spectroscopic studies (ESEEM, ENDOR) allow us to probe structure local to paramagnetic catalytic intermediates that are not accessible through crystallography. Our current work involves the role of semiquinone intermediates, the pathway for H⁺ release from catalytic sites through water chains, and electron transfer across the dimer interface that complicates the simple monomeric Q-cycle mechanism. We are studying these using a variety of biophysical, biochemical, computational and molecular engineering approaches. The project also involve several local and international collaborative projects.

A more esoteric interest comes from attempts to quantify global fluxes in the biosphere. This introduces questions about information fluxes, the role of semantic content and its thermodynamic status, leading to deeper philosophical interests.

Representative Publications

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

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.

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

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

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

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

Crofts, A.R. and Rose, S. (2007) Marcus treatment of endergonic reactions: A commentary. Biochim. Biophys. Acta 1767, 1228–1232 (PMC2238675)

Crofts, A.R. (2007) Life, information, entropy, and time. Complexity, 13, 14-50 (PMC2577055)

Crofts, A.R., Holland, J.T., Victoria, D., Kolling D.R.J., Dikanov, S.A., Gilbreth, R., Lhee, S., Kuras, R., and Guergova-Kuras, M. (2008) The Q-cycle reviewed: How well does a monomeric mechanism of the bc₁ complex account for the function of a dimeric complex? Biochim. Biophys. Acta, 1777, 1001-1019 (PMC2578832)

Kolling, D.R.J., Samoilova, R.I., Shubin, A.A., Crofts, A.R. and Dikanov, S. A. (2009) Proton Environment of Reduced Rieske Iron-Sulfur Cluster Probed by Two-Dimensional ESEEM Spectroscopy. J. Phys. Chem. 113, 653-667 (PMC2773023)

Dikanov, S.A., Samoilova, R.I., Kappl, R., Crofts, A.R. and Hüttermann, J. (2009) The reduced [2Fe-2S] clusters in adrenodoxin and Arthrospira platensis ferredoxin share spin density with protein nitrogens, probed using 2D ESEEM. Phys. Chem. Chem. Phys. 11, 6807-6819 (PMC2773023)

Lhee, S., Kolling, D. R., Nair, S. K., Dikanov, S. A. and Crofts, A. R. (2010) Modifications of protein environment of the [2Fe-2S] cluster of the bc₁ complex: Effects on the biophysical properties of the Rieske iron-sulfur protein and on the kinetics of the complex. J. Biol. Chem. 285, 9233-9248 (PMC2838342)

Complete Publications List