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It Is Chemistry but Not Your Grandfather’s Chemistry Aziz Sancar* University of North Carolina School of Medicine, Chapel Hill, North Carolina 27516, United States
Dongping Zhong The Ohio State University, Columbus, Ohio 43210, United States
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n recent years, the awards of Nobel Prize in Chemistry has generated discontent among some chemists who complain that more and more the Chemistry Prize is given to biologists, with expressions like “But, is this chemistry?” With all due respect to “chemist” colleagues who ask the question, or the rhetorical comment “Is this chemistry?”, when the Chemistry Prize is given for research in biological chemistry, our answer is “Do your homework before asking that question.” We present the following example to illustrate the point. The Nobel Prize in Chemistry 2015 was given to three scientists for their work on “mechanistic studies of DNA repair” that includes DNA repair by photolyase. This enzyme uses photons as a cosubstrate to break two C−C bonds in one of the most celebrated structures in organic chemistry textbooks, the cyclobutane ring. The enzyme binds to cyclobutane pyrimidine dimers (CPD) induced in DNA by UV and flips out the CPD from within the duplex into the active site cavity to produce a rather stable E−S complex. Catalysis is initiated by absorption of a photon (350−450 nm). The enzyme has two chromophore/cofactors: methenyltetrahydrofolate (MTHF) [in some photolyases 8-hydroxy 5-deazaflavin (HDF)] and two-electron reduced and deprotonated flavin adenine dinucleotide (FADH−). The blue light photon is absorbed by MTHF (or HDF), which transfers the excitation energy to FADH− by a Forster resonance energy transfer mechanism; the FADH−* then transfers an electron to the CPD, generating a radical pair, FADH• and CPD•−. The C5−C5 and C6−C6 bonds of CPD•− are cleaved in a stepwise reaction, the back electron transfer restores the catalytic cofactor to the FADH− form, the repaired pyrimidine dinucleotide flips out from the enzyme active site and into the double helix, and the enzyme dissociates from repaired DNA (Figure 1).1−4 The enzyme was purified by LC and FPLC (protein chemistry) and analyzed by UV−vis and fluorescence spectroscopy, and the cofactors were isolated by TLC and HPLC and identified by absorption and fluorescence properties and MS and NMR (analytical chemistry). The redox state of the flavin cofactor was determined by EPR in vivo and in vitro. The structures of the enzyme and enzyme−substrate complex were determined by EM and X-ray crystallography (structural chemistry). The enzyme−substrate binding was analyzed by conventional thermodynamic and kinetic approaches (physical chemistry). The structural and kinetic factors for the preference of cis,syn-CPD over trans,syn-CPD by the enzyme were determined (stereochemistry). The mechanism by which the enzyme circumvents the Woodward−Hoffmann rule for conservation of orbital symmetry (HOMO, LUMO, SOMO) in cleavage of chemical bonds was explained (quantum © 2017 American Chemical Society
Figure 1. Photolyase Structure and Photocycle. The active site of the enzyme with the cyclobutane pyrimidine dimer (CPD) and the elementary steps in cleavage of the cyclobutane ring are shown. The photoantenna (HDF) absorbs a blue light photon transferring the excitation energy to FADH− through resonance energy transfer. This is followed by electron transfer from FADH− to CPD to effect the repair reaction that encompasses 10 elementary steps including 7 electron transfer processes as highlighted in the photocycle.
chemistry). The stepwise C5−C5 and C6−C6 bond cleavage was probed by measuring the deuterium isotope effect on reaction kinetics (transition state chemistry). The enzyme was utilized to electrochemically monitor DNA repair (electrochemistry). Using ultrafast absorption and fluorescence spectroscopy, bond cleavage and re-formation were observed in real time and at femtosecond resolution (femtochemistry). It was found that forward electron transfer is in the Marcus normal region and involves both electron tunneling and electron hopping and that the return of the electron from the substrate to the flavin cofactor following bond cleavage is in the Marcus inverted region (photochemistry). Characterization of the enzyme and its reaction mechanism were accomplished by using the following methods: conventional column chromatography, FPLC, HPLC, TLC, MS, NMR, EPR, EM (electron microscopy), X-ray crystallography, UV−vis spectroscopy, fluorescence spectroscopy (conventional), flash photolysis (millisecond), cyclic voltammetry, Photo-CIDNP, ultrafast spectroscopy (absorption and fluorescence up-conversion), and the list goes on. Is this truly chemistry? In the immortal words of ESPN, “C’mon Man!” Received: December 15, 2016 Published: January 10, 2017 1
DOI: 10.1021/acs.biochem.6b01262 Biochemistry 2017, 56, 1−2
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Biochemistry
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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REFERENCES
(1) Sancar, A. (1994) Structure and function of DNA photolyase. Biochemistry 33, 2−9. (2) Sancar, A. (2003) Structure and function of DNA photolyase and cryptochrome blue-light photoreceptors. Chem. Rev. 103, 2203−2237. (3) Zhong, D. (2015) Electron transfer mechanisms of DNA repair by photolyase. Annu. Rev. Phys. Chem. 66, 691−715. (4) Zhang, M., Wang, L., Shu, S., Sancar, A., and Zhong, D. (2016) Bifurcating electron-transfer pathways in DNA photolyases determine the repair quantum yield. Science 354, 209−213.
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DOI: 10.1021/acs.biochem.6b01262 Biochemistry 2017, 56, 1−2
