[27 Defining Redox State of XRay Crystal Structures by Single Crystal Ultraviolet Visible Microspectrophotometiy

By Carrie M. Wilmot, Tove Sjögren, Gunilla H. Carlsson, Gunnar I. Berglund, and Janos Hajdu

Introduction

Exciting results have been emerging from the field of single-crystal X-ray crystallography, giving unprecedented detail of freeze-trapped reaction intermediates from important classes of macromolecules that contain chromophores1-10 (for reviews see Refs. 11-13). The reason for the current excitement is that these structures have been coupled with single-crystal UV-visible microspectrophotometry. This has defined the distinct catalytic intermediates present in the crystal structures, allowing the correlation of electronic transitions with the observed structural transitions. Of particular note is that many of these structures have been generated "on the fly" during kinetic turnover in the crystal. Most enzymatic reactions proceed through distinct catalytic intermediates that, under favorable conditions, may accumulate transiently in the crystal during turnover. In some cases, the physical constraints of the contacts within crystals may also lead to a significant slowing of

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METHODS IN ENZYMOLOGY, VOL. 353

Copyright 2002, Elsevier Science (USA).

All rights reserved. 0076-6879/02 $35.00

the reaction at certain points along the pathway where conformational changes are required. This can lead to a transient build-up of spectrally distinct intermediates in the crystal that can be trapped by flash freezing in liquid nitrogen, allowing a complete single-crystal data set to be collected to the highest possible resolution at a later time. Similar build-up of intermediates may be achieved by altering the pH, temperature, or the solvent environment around the protein in the crystal, or by producing engineered variants that build up an intermediate of interest.

This chapter focuses on the technical considerations required to carry out UV-visible microspectroscopy of single crystals.

Historical Problems

The large solvent channels found in macromolecular crystals have been allowing crystallographers to soak drugs/products/substrate analogs into enzyme crystals for decades. These single-crystal X-ray structures of enzyme complexes have aided our understanding of function. Although these can give insight to the chemical groups involved and suggest mechanisms, the structures represent beginning or end points of the reaction. Critics also suggested that the constraints of crystal contacts could lead to kinetically artificial structures, not only via kinetic dead-ends, but by favoring a minor reaction pathway over the major pathway observed in solution. Particularly in redox enzymes the reactions often lead to forms that are X-radiation sensitive. In this case the oxidation state of redox centers may be affected by X-rays during data collection. Figure 1 shows spectral changes in a crystal of the compound III form of horseradish peroxidase during the collection of 90° of X-ray data. These results demonstrate a general problem with redox proteins; without spectral controls it is practically impossible to obtain reliable structures for oxidized catalytic intermediates, because electrons liberated during X-ray exposure alter the oxidation state of the redox center in the active site. Absorption spectroscopic studies of single crystals of numerous heme enzymes show that, contrary to general belief, the redox state of the heme iron is affected by electrons liberated in the sample even at the shortest X-ray wavelengths.14'15 In addition, the medium surrounding the crystal can influence X-ray sensitivity. The original structure of an "oxidized" ribonucleotide reductase had metal ligation identical to that of the reduced enzyme. Glycerol, a good electron hole stabilizer, was present in the crystal mother liquor, and had facilitated reduction of the iron in the X-ray beam. Eventually the structure of the oxidized enzyme was solved by excluding glycerol from the crystal-stabilizing solution.16 As radiation damage is a major concern in the structure determination of redox intermediates of proteins, it is useful to understand the basis for this.

14 G. I. Berglund, G. H. Carlsson, A. T. Smith, H. Szoke, A. Henriksen, and J. Hajdu, Nature (London), in press (2002).

15 B. Ziaja, D. van der Spoel, A. Szoke, and J. Hajdu, Phys. Rev. B64, 214104 (2001).

16 M. Eriksson, A. Jordan, and H. Eklund, Biochemistry 37,13359 (1998).

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