Metalloprotein in solution

Figure 4 A schematic representation of the experimentai approach for time-resoived XAS measurements. XAS provides local structural and electronic information about the nearest coordination environment surrounding the catalytic metal ion within the active site of a metalloprotein in solution. Spectral analysis of the various spectral regions yields complementary electronic and structural information, which allows the determination of the oxidation state of the X-ray absorbing metal atom and precise determination of distances between the absorbing metal atom and the protein atoms that surround it. Time-dependent XAS provides insight into the lifetimes and local atomic structures of metal-protein complexes during enzymatic reactions on millisecond to minute time scales, (a) The drawing describes a conventional stopped-flow machine that is used to rapidly mix the reaction components (e.g., enzyme and substrate) and derive kinetic traces as shown in (b). (b) The enzymatic reaction is studied by pre-steady-state kinetic analysis to dissect out the time frame of individual kinetic phases, (c) The stopped-flow apparatus is equipped with a freeze-quench device. Sample aliquots are collected after mixing and rapidly froze into X-ray sample holders by the freeze-quench device, (d) Frozen samples are subjected to X-ray data collection and analysis.

Concentrating on metalloenzymes, we have developed a strategy based on stopped flow X-ray absorption spectroscopy (XAS) to elucidate in detail the molecular mechanisms at work during substrate turnover (Fig. 4). Importantly, XAS provides local stmctural and electronic information about the nearest coordination environment surrounding the catalytic metal ion within the active site of a metalloprotein in solution. When the X-rays hit a sample, the electromagnetic radiation interacts with the electrons bound in the metal atom. The radiation can be scattered by these electrons, or it can be absorbed, thereby exciting... 

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