The bond valence sum (BVS) method is further applied to metalloenzyme active sites. When a particular coordination model is assumed, the BVS method allows for oxidation states of metal ions in metalloproteins to be determined from metal-ligand bond distances measured using extended X-ray absorption fine structure (EXAFS) analysis. Thus, the BVS can be used to determine the compatibility between a given coordination model and a particular oxidation state. A new procedure for calculating ro values on which BVS's are based is presented. This procedure allows for calculation of ro values on heteroleptic complexes and was used to determine a new set of ro distances using complexes that more closely model the active sites of interest. In particular, the new distances allow for calculations involving vanadium, molybdenum, and nickel. New calculations using EXAFS data on CO dehydrogenase, NiFe hydrogenase, manganese catalase, sulfite oxidase, MoFe nitrogenase, and VFe nitrogenase are presented. The interplay between oxidation state and coordination geometry can be quantitatively assessed using the BVS method.Determining the structures of metal ion binding sites is critical in understanding the mechanism of action of metalloenzymes. Extended X-ray absorption fine structure (EXAFS) provides a means for determining the metal-ligand bond lengths for a given metal ion in a protein. l-3 X-ray absorption near-edge spectroscopy (XANES) can also provide some information on the oxidation stateof the metal Recently, we reportedon theapplication of the bond valence sum (BVS) method to the analysis of metalligand bond lengths determined by EXAFS in metalloenzymes.5 In particular, when a structural model is deduced from EXAFS and other spectroscopic methods, the BVS can be used to confirm the compatibility of that model with a given oxidation state of the metal ion. Conversely, if the oxidation state is known, BVS can be used to test structural models. We have demonstrated that this method is effective in a variety of mononuclear and polynuclear metal ions in a wide range of oxidation states for a series of metalloenzymes.Bond valences (s) are calculated according to eq 1, where B *Abstract published in Advance ACS Abstracts, August 15, 1993. Koningsberger, D. C., Prins, R., Eds.; X-ray Absorption: Principles, Applications, Techniques of EXAFS, SEXAFS, and XANES; John Wiley and Sons: New York, 1988. Stavrapoulos, P.; Muetterties, M. C.; Carrib, M.; Holm, R. H. J. Am. Chem. SOC. 1991, 113, 8485. Bastian, N . R.; Diekert, G.; Niederhoffer, E. C.; Teo, B.-K.; Walsh, C. T.; Orme-Johnson, W. H. J. Am. Chem. SOC. 1988, 110, 5581. Tan, G. 0.; Ensign, S. A.; Ciurli, S.; Scott, M. J.; Hedman, B.; Holm, R. H.; Ludden, P. W.; Korzun, 2. R.; Stephens, P. J.; Hodgson, K. 0. Proc. Narl. Acad. Sci. U.S.A. 1992, 89, 4427. Baidya, N.; Olmstead, M.; Mascharak, P. K. Inorg. Chem. 1991, 30, 929. Baidya, N.; Olmstead, M. M.; Mascharak, P. K. J . Am. Chem. SOC. 1992, 114, 9666. The Bioinorganic Chemistry ofNickel; Lancaster, L. R., Jr., Ed.; VCH Publishers...

All higher life forms use oxygen and respiration as their primary energy source. The oxygen comes from water by solar-energy conversion in photosynthetic membranes. In green plants, light absorption in photosystem II (PSII) drives electron-transfer activation of the oxygen-evolving complex (OEC). The mechanism of water oxidation by the OEC has long been a subject of great interest to biologists and chemists. With the availability of new molecular-level protein structures from X-ray crystallography and EXAFS, as well as the accumulated results from numerous experiments and theoretical studies, it is possible to suggest how water may be oxidized at the OEC. An integrated sequence of light-driven reactions that exploit coupled electron-proton transfer (EPT) could be the key to water oxidation. When these reactions are combined with long-range proton transfer (by sequential local proton transfers), it may be possible to view the OEC as an intricate structure that is "wired for protons".

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Indium tin oxide electrodes were modified with DNA, and the guanines in the immobilized nucleic acid were used as a substrate for electrocatalytic oxidation by Ru(bpy)3(3+) (bpy = 2,2'-bipyridine). Nucleic acids were deposited onto 12.6-mm2 electrodes from 9:1 DMF/water mixtures buffered with sodium acetate. The DNA appeared to denature in the presence of DMF, leading to adsorption of single-stranded DNA. The nucleic acid was not removed by vigorous washing or heating the electrodes in water, although incubation in phosphate buffer overnight liberated the adsorbed biomolecule. Acquisition of cyclic voltammograms or chronoamperomograms of Ru(bpy)3(2+) at the modified electrodes produced catalytic signals indicative of oxidation of the immobilized guanine by Ru(III). The electrocatalytic current was a linear function of the extent of modification with a slope of 0.5 microA/pmol of adsorbed guanine; integration of the current-time traces gave 2.2+/-0.4 electrons/guanine molecule. Use of long DNA strands therefore gave steep responses in terms of the quantity of adsorbed DNA strand. For example, electrodes modified with a 1497-bp PCR product from the HER-2 gene produced detectable catalytic currents when as little as 550 amol of strand was adsorbed, giving a sensitivity of 44 amol/mm2.

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The electrocatalytic oxidation of guanine in DNA and oligonucleotides by Ru(bpy) 3 3+/2+ was investigated using cyclic voltammetry (CV) and chronoamperometry (CA) (bpy ) 2,2′-bipyridine). Oxidation of Ru(bpy) 3 2+ to the Ru(III) form at tin-doped indium oxide (ITO) electrodes in the presence of DNA produces catalytic current due to the oxidation of guanine by Ru(III). CA traces of Ru(bpy) 3 2+ with calf thymus DNA at high salt concentration (50 mM sodium phosphate + 700 mM NaCl) give a single kinetic process with a rate constant of 3500 ( 300 M -1 s -1 that is independent of DNA concentration and similar to values determined previously by fitting CV data under the same conditions. Under low salt conditions (50 mM sodium phosphate + 0 mM NaCl), the CA data show two linear regions that give rate constants of 2.7 × 10 4 and 6 × 10 5 M -1 s -1 . Digital simulation of CV data at low salt requires a careful accounting for the binding of the metal complex to the DNA polyanion, which can be accomplished using binding constants that are independently determined. This analysis gives rate constants that are independent of DNA concentration and range from 2.3 × 10 5 to 1.4 × 10 6 M -1 s -1 as the scan rate is increased from 25 to 250 mV/s. The variation in rate constant with scan rate can be attributed to the two kinetic processes observed in the CA results. Resolution of Ru(bpy) 3 2+ into the ∆ and Λ stereoisomers showed that the two kinetic processes were not due to the stereoisomerism. Satisfactory fitting of the CV data requires addition of a second electron-transfer step from the oxidized guanine to Ru(III); this rate constant was always less than 1% of the rate constant for the first homogeneous electron transfer. In addition, the fitting at low salt requires accounting for the density of guanines in the DNA sequence. Calf thymus DNA is 20% guanine; however, the fitting shows that binding of the mediator by at least 60% of the nucleotides produces catalytic turnover. Oligonucleotides containing a single guanine gave similar rate constants to those observed by CV and CA on calf thymus DNA, and the fitting suggested that binding of the mediator by 5-10 of the 30 nucleotides (defined as "active binding sites") in the oligomer produced catalyst cycling. Thus, the electron is able to transfer to a mediator that is bound in a region that spans 2.5-5 base pairs and contains the oxidized guanine. The number of "active binding sites" increased predictably with the number of guanines in the sequence, ranging from 15% to 33% of the total nucleotides for a 15-mer duplex with one guanine to 75-100% for a 15-mer duplex with six guanines. Decreasing the salt concentration enhances the catalytic current both by increasing the number of active binding sites by a factor of 5-10 and by increasing the intrinsic oxidation rate by an order of magnitude.

The cyclic voltammetry of Ru(bpy)3 2+ in the presence of calf thymus DNA has been studied. Theoretical simulations using DigiSim were performed for the voltammetry of the metal complex in the presence of DNA where the only interaction between the metal complex and DNA was electrostatic binding to the polyanion. The expected binding isotherm was obtained from the simulated voltammetry with input affinities similar to that of Ru(bpy)3 2+. The expected binding isotherm was not obtained for simulations with high affinities (>106 M-1) expected for intercalating complexes that exhibit neighbor exclusion, because the commercially available version of DigiSim treats only simple equilibria and cannot treat exclusion of binding to adjacent sites. Simulations were then performed for the case where the +3 state of the metal complex oxidizes the guanine base in DNA in a catalytic mechanism. The dependence of i cat/i d on scan rate and the second-order rate constant for the homogeneous chemical step was determined for the conditions where the metal complex does not bind to DNA, such as at high salt concentration. Under these conditions, there is an optimum scan rate where the catalytic current depends steeply on the homogeneous rate constant, allowing for the most accurate determinations in fitting experimental voltammograms. These considerations were applied to fitting cyclic voltammograms for the case of no DNA binding (high salt) and weak, but significant, DNA binding (50 mM salt). The rate of homogeneous electron transfer from the guanine nucleobase to the metal complex was 10 times faster in the low salt case, indicating a shorter electron-transfer distance and a more intimate association of the metal complex with the DNA.