Oj

Semiquinone intermediate

Fig. 5. Spectral changes spaced 1 min apart during enzyme catalysis in crystals of E. coli amine oxidase. Spectra were collected every 20 sec, and every third spectrum is shown.

Steady state

Semiquinone intermediate

- Oxidized E. coli amine oxidase soo

Wavelength (nm)

Fig. 5. Spectral changes spaced 1 min apart during enzyme catalysis in crystals of E. coli amine oxidase. Spectra were collected every 20 sec, and every third spectrum is shown.

ts3 vl

Fig. 6. The rate-determining intermediate in crystals of E. coli amine oxidase (PDB code 1D6Z) [C. M. Wilmot, J. Hajdu, M. J. McPherson, P. F. Knowles, and S. E. V. Phillips, Science 286,1724 (1999)] corresponding to species 4 in Fig. 4. Dashed lines indicate key hydrogen bonds and van der Waals interactions. (Reprinted with permission, copyright 1999 American Association for the Advancement of Science.)

Fig. 6. The rate-determining intermediate in crystals of E. coli amine oxidase (PDB code 1D6Z) [C. M. Wilmot, J. Hajdu, M. J. McPherson, P. F. Knowles, and S. E. V. Phillips, Science 286,1724 (1999)] corresponding to species 4 in Fig. 4. Dashed lines indicate key hydrogen bonds and van der Waals interactions. (Reprinted with permission, copyright 1999 American Association for the Advancement of Science.)

Other Hardware for Single-Crystal Optical Microspectrophotometry

Single-crystal microspectrophotometers have been adapted so that the optics can be mounted directly onto the goniometers of X-ray cameras. This allows the crystal to be monitored during data collection for any redox changes in the beam.14,19

A flow system that keeps a moist atmosphere around a naked crystal bathed in cryoprotectant in a loop has been developed. A cryostream can be set up with the system, with the cold stream blocked from the crystal. Substrate can be added directly to the loop, allowing kinetics to be monitored directly, and the cold stream can be unblocked to freeze the crystal instantaneously at the point of maximal build-up of the desired intermediate, and enable X-ray data collection. In addition, this system allows the atmosphere around the crystal to be controlled, for example, at a defined oxygen concentration.

By linking the single-crystal microspectrophotometer stage (Fig. 2) with an infrared (IR) source and detector using appropriate optics, single-crystal Fourier transform infrared (FTIR) spectroscopy can be performed.25 The identity of small ligands observed in protein crystal structures can then be determined by difference IR.26-27 In principle, there is no reason why the microspectrophotometers cannot

25 J. M. Hadden, D. Chapman, and D. C. Lee, Biochim. Biophys. Acta 1248, 115 (1995).

26 J. C. Fontecilla-Camps, M. Frey, E. Garcin, C. Hatchikian, Y. Montet, C. Piras, X. Vernede, and A. Volbeda, Biochimie 79,661 (1997).

27 H. Khachfe, M. Mylrajan, and J. T. Sage, Cell. Mol. Biol. 44, 39 (1998).

also be adapted for fluorescence measurements, while single-crystal micro-Raman spectroscopy was demonstrated in the early 1990s.28

This is an exciting time in redox biochemistry: flash freezing of crystals, coupled with supporting spectroscopies, is allowing "snapshots" from the catalytic cycles of enzymes, obtained by single-crystal X-ray crystallography, to be accurately placed along the reaction pathway, giving structural insight into the controlling factors of biological catalysis.

28 G. Smulevich and T. G. Spiro, Methods Enzymol. 226, 397 (1993).

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