Metalloproteins in solution

In metalloproteins, the paramagnet is an inseparable part of the native biomacromolecule, and so anisotropy in the metal EPR is not averaged away in aqueous solution at ambient temperatures. This opens the way to study metalloprotein EPR under conditions that would seem to approach those of the physiology of the cell more closely than when using frozen aqueous solutions. Still the number of papers describing metalloprotein bioEPR studies in the frozen state by far outnumbers studies in the liquid state. Several additional theoretical and practical problems are related to the latter (1) increased spin-lattice relaxation rate, (2) (bio)chemical reactivity, (3) unfavorable Boltzmann distributions, (4) limited tumbling rates, and (5) undefined g-strain. 

Most biochemically relevant high-spin systems have such short 7j-relaxation times that their EPR is broadened beyond detection at ambient temperatures. An exception is the class of S = 5/2 Mn systems with D hx. Also, S = 7/2 Gd 1-based MRI shift reagents exhibit readily detectable room-temperature EPR spectra. Otherwise, aqueous-solution transition ion bioEPR is limited to complexes of S = 1/2 metals, in particular Cu , and to a lesser extent VIV02+, NiIn, Ni1, Mov, and Wv. Cupric is the stable oxidation state of biological copper under aerobic conditions, however, the other metals are stable as Vv, Ni , MoVI, and WVI, and, therefore, the other oxidation states associated with S = 1/2 paramagnetism may exhibit oxidative or reductive reactivity and may thus require specific experimental precautions such as strict anaerobicity over the course of the EPR experiment. 

FIGURE 10.6 Comparison of solid-state and liquid-state spectra from a copper protein. The figure illustrates shifts in apparent gz and Az-values of the S = 1/2 and I =3/2 spectrum from Cu11 in bovine superoxide dismutase as a function of the surrounding medium. Top trace frozen aqueous solution at 60 K middle trace frozen water/glycerol (90/10) solution at 60 K bottom trace aqueous solution at room temperature. (Modified from Hagen 1981.) 

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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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