Spectroscopic and thermodynamic comparisons of Escherichia coli DNA photolyase and Vibrio cholerae cryptochrome 1.
Escherichia coli DNA photolyase and cryptochrome 1 isolated from Vibrio cholerae, a member of the CRY-DASH family, are directly compared using a variety of experimental methods including UV-vis and Raman spectroscopy, reduction potential measurements, and isothermal titration calorimetry. The semiquinone form of the cryptochrome has an absorption spectrum that is red-shifted from that of the photolyase, but the Raman spectrum indicates that the FAD binding pocket is similar to that of photolyase. The FADH(-)/FADH* reduction potential of the cryptochrome is significantly higher than that of the photolyase at 164 mV vs NHE, but it also increases upon substrate binding (to 195 mV vs NHE), an increase similar to what is observed in photolyase. The FADH(-)/FADH* reduction potential for both proteins was found to be insensitive to ATP binding. Isothermal titration calorimetry found that photolyase binds tighter to substrate (K(A) approximately 10(5) M(-1) for photolyase and approximately 10(4) M(-1) for cryptochrome 1), and the binding constants for both proteins were slightly sensitive to oxidation state. Based upon this work, it appears that this cryptochrome has significant spectroscopic and electrochemical similarities to CPD photolyase. The thermodynamic cycle of the enzymatic repair in the context of this work is discussed.
Sokolowsky
Kathleen
K
Department of Chemistry, Hugel Science Center, Lafayette College, Easton, Pennsylvania 18042, USA.
Newton
Maire
M
Lucero
Carlos
C
Wertheim
Bradley
B
Freedman
Jaryd
J
Cortazar
Frank
F
Czochor
Jennifer
J
Schelvis
Johannes P M
JP
Gindt
Yvonne M
YM
D002151:Calorimetry; D056931:Cryptochromes / Q000737:chemistry*; D004255:Deoxyribodipyrimidine Photo-Lyase / Q000737:chemistry*; D004926:Escherichia coli / Q000201:enzymology*; D005182:Flavin-Adenine Dinucleotide / Q000737:chemistry; D010084:Oxidation-Reduction; D011485:Protein Binding; D013056:Spectrophotometry, Ultraviolet; D013059:Spectrum Analysis, Raman; D013816:Thermodynamics; D014734:Vibrio cholerae / Q000201:enzymology*
D056931:Cryptochromes; D005182:Flavin-Adenine Dinucleotide; D004255:Deoxyribodipyrimidine Photo-Lyase