We have used a new approach to the dynamics of hydrolytic metalloenzyme catalysis based on investigations of both external solvent viscosity effects and kinetic 'H isotope effects. The former reflects solvent and protein dynamics, and the nuclear reorganization distribution among damped protein motion and intramolecular frictionfree nuclear motion. The isotope effect represents proton tunnelling and reorganization in the hydrogen bond network around the active site.We illustrate the approach by new spectrophotometric and pH-titration data for carboxypeptidase-A-catalyzed benzoylglycyl-t-phenyllactate hydrolysis. This substrate exhibits both a significant inverse fractional power law viscosity dependence over wide ranges controlled by glycerol and sucrose, and a kinetic 'H isotope effect of 1.65. The analogous benzoylglycylphenylalanine hydrolysis has a smaller isotope effect (1.3) and no viscosity dependence. Viscosity variation has no effect on the CD spectra in the 180-240-nm range. In terms of stochastic chemical rate theory, the data correspond to an enzyme-peptide substrate complex with a 'tight' structure protected from the solvent. In comparison, the enzyme-ester substrate complex is 'softer', strongly coupled to the solvent, and the rate-determining step is accompanied by proton transfer or by substantial reorganization in the hydrogen bonds near the active site.Protein dynamics is spanned by broad time ranges extending from sub-picoseconds to milliseconds, and even seconds [l-41. The fastest limits are represented by lattice-like atomic motion [ l -31, the slowest by denaturation or subunit association, where large protein segments are rearranged. . This points towards solvent-mediated strong damping of the conformational relaxation. These modes must therefore be associated with the vibrationally dispersive solvent relaxation properties. Among these, viscosity has come to stand out prominently.Low-frequency dielectric 'loss' is a universal feature of correlated nuclear motion in liquids and disordered solids [29], associated with diverse properties such as dielectric relaxation, viscoelasticity, etc., and including conformational protein motion. Relaxation times are determined by the thermalization rates along the assembly of dissipative coordinates, subsequent to external perturbations. The dissipative, o r frictional features are associated with interaction or collision with the nuclear fragments, and collectively grouped into what is denoted as a microviscosity function [30]. This function can be dominated by external solvent collisional properties for processes involving protein surface fragments since liquid 'hole' formation in the solvent near the protein surface must precede the surface group motion. The microviscosity, however, differs from the bulk 'kinematic' viscosity, even though both quantities represent intermolecular collisions, liquid hole formation, etc. [30]. The external solvent dependence is thus

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The 52-residue Desulfovibrio gigas rubredoxin peptide chain has been synthesized and a procedure for chain folding around iron(I1) developed. The folded, stable synthetic rubredoxin can be subjected to purification, and reversibly oxidized and reduced. Ultraviolet/visible absorption and CD spectra of both forms show all the same features as native D. gigas rubredoxin, and the symmetric and asymmetric Fe-S stretching bands in the resonance Raman spectrum can be identified. In addition, the matrix-assisted laser desorption mass spectrum of a peptide sample exposed to trace amounts of iron is dominated by a peak at 5735Da very close to the value for the calculated molecular mass. Details in the ultraviolethisible bandshape and mass spectrum, however, indicate remaining impurities. In comparison, a previously synthesized 25-residue rubredoxin fragment with the non-conserved positions 13-35 and 51 -52 omitted and Val5 -Glu50 anchored via glycine folds gives the correct molecular mass and ultraviolethisible spectrum, but is much more labile than the 52-residue protein. This shows that non-conserved residues are crucial in protein folding and that chemical metalloprotein synthesis offers alternative prospects to microbiological protein engineering.Total chemical synthesis of small, naturally occurring metalloproteins is becoming feasible, as indicated by recent studies of Clostridium pasteurianum ferredoxin [I], racemic Desulfovibrio desulfuricans rubredoxin [2], and horse heart cytochrome c [3]. While presently not competitive with microbiological methods, chemical metalloprotein synthesis is a potential powerful alternative to site-modification of proteins. These techniques can be used as tools for mapping physical and chemical properties of the protein, and factors important in maintaining the folded structure. Chemical metalloprotein synthesis also offers an additional perspective, namely that the peptide chain can both be modified and reduced in size. Metal complexes with peptide chain ligands large enough to fold but smaller than the chains in native proteins can thus be synthesized. This method would offer extremely efficient approaches to structural and functional metalloprotein mapping once the preparative procedures are powerful enough to provide the appropriate amounts of protein. The prospects are illustrated by a recently synthesized 61-residue protein with a novel three-strand p fold termed minibody [4]. Elements responsible for the stabilization due to folding for this protein, which forms a template for the heavy-chain domain of immunoglobin, were mapped and a binding site consisting of three histidine residues, following Correspondence to J. Ulstrup, Chemistry Department A, Building 207, The Technical University of Denmark, DK-2800 Lyngby, DenmarkAbbreviations. Fmoc , N-9-fluorenylmethox ycarbon yl ; MALDI-MS, matrix-assisted laser desorption ionization mass spectrometry. a metal-selectivity pattern similar to carbonic anhydrase, was engineered into the protein.Biomimetic metal complexes of peptides wi...

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