Isothermal titration calorimetry (ITC) was used to quantify the thermodynamics of Pb(2+) and Zn(2+) binding to metallothionein-3 (MT-3). Pb(2+) binds to zinc-replete Zn7MT-3 displacing each zinc ion with a similar change in free energy (ΔG) and enthalpy (ΔH). EDTA chelation measurements of Zn7MT-3 and Pb7MT-3 reveal that both metal ions are extracted in a tri-phasic process, indicating that they bind to the protein in three populations with different binding thermodynamics. Metal binding is entropically favoured, with an enthalpic penalty that reflects the enthalpic cost of cysteine deprotonation accompanying thiolate ligation of the metal ions. These data indicate that Pb(2+) binding to both apo MT-3 and Zn7MT-3 is thermodynamically favourable, and implicate MT-3 in neuronal lead biochemistry.

Conditions have been developed for the comproportionation reaction of Cu2+ and copper metal to prepare aqueous solutions of Cu+ that are stabilized from disproportionation by MeCN and other Cu+-stabilizing ligands. These solutions were then used in ITC measurements to quantify the thermodynamics of formation of a set of Cu+ complexes (CuI(MeCN)3+, CuIMe6Trien+, CuI(BCA)23−, CuI(BCS)23−), which have stabilities ranging over 15 orders of magnitude, for their use in binding and calorimetric measurements of Cu+ interaction with proteins and other biological macromolecules. These complexes were then used to determine the stability and thermodynamics of formation of a 1 : 1 complex of Cu+ with the biologically important tri-peptide glutathione, GSH. These results identify Me6Trien as an attractive Cu+-stabilizing ligand for calorimetric experiments, and suggest that caution should be used with MeCN to stabilize Cu+ due to its potential for participating in unquantifiable ternary interactions.

An enzymatic system for light-driven hydrogen generation has been developed throughc ovalent attachment of ar uthenium chromophore to nickel-substituted rubredoxin (NiRd).The photoinduceda ctivity of the hybrid enzymei ss ignificantly greater than that of at wo-component system and is strongly dependent on the positiono ft he ruthenium phototriggerr elative to the active site, indicating ar ole for intramolecular electron transfer in catalysis. Steady-state and time-resolved emission spectra reveal ap athway for rapid, directq uenching of the ruthenium excited state by nickel, butl ow overall turnover numberssuggest initial electron transfer is not the rate-limiting step. This approach is ideally suited for detailed mechanistic investigationso fc atalysis by NiRd and other molecular systems, with implications for generation of solar fuels.Hydrogeni sc onsidered to be ac lean, renewable alternative to carbon-based fuels. However, generating hydrogen through traditional methods such as electrolytic water splitting can be costly and energy intensive, thus overriding the advantages of this supposedly sustainable fuel. [1][2][3] In contrast to anthropogenic approaches, biological systems have evolvedt op roduce hydrogen under mild conditions by using proteins called hydrogenases. [4,5] Although theseh ighly efficient enzymes suffer from extreme fragilityu nder atmospheric conditions, precluding general application, they have inspired the development of chemically diverse hydrogenase models. [6][7][8][9][10][11][12][13][14][15][16][17][18] Of particulari nterest is the design of systemst hat can be driven by light for the generation of so-called "solar fuels". [19][20][21][22][23][24][25] We have shown that nickel-substituted rubredoxin (NiRd),asmall electron-transfer protein that naturally binds iron in at etrahedral tetrathiolate coordination motif, mimics the structure and function of the redox-active site in the [NiFe] hydrogenases. The hydrogenevolvinga ctivity of this system has previously been characterized by using solution-based photochemical and electrocatalytic assays. [26] In this study,aruthenium chromophore was covalently attachedt oN iRd to directlyr educe the active site for light-initiated catalysis. Attachment at different locations aroundt he protein surface reveals as trong distance dependence for hydrogen evolution activity,a nd time-resolved emission studies were used to investigate the efficiency of electron transfer between the ruthenium and nickel centers. Low overall turnover numbers across all of the NiRd variants suggest initial electron transfer is not the rate-limiting step in catalysis.The ruthenium chromophore [Ru II (2,2'-bipyridine) 2 (5,6-epoxy-5,6-dihydro-[1,10]-phenanthroline)] 2 + was synthesized as previously reported and bound to the sulfhydryl group of af ree cysteiner esidue. [27] Following purification,t he labeling efficiency was found to be 90 AE 5% by optical absorption (see the Supporting Information, Figure S1). MALDI-TOF mass spectrometry confirmed this yield, showing ...