| AUTHOR |
TITLE |
Davidson, V. L.,
Jones, L. H. |
Electron Transfer from Copper to Heme within the Methylamine Dehydrogenase-Amicyanin-Cytochrome c-551i Complex
Biochemistry; 1996; 35(25); 8120-8125. (Article) |
Experimental Procedures
An On-Line Instruments (OLIS, Bogart, GA) RSM1000 stopped-flow rapid scanning spectrophotometer was used for kinetic measurements. All experiments were performed in 0.01 M potassium phosphate, pH 7.5, as described previously (Davidson & Jones, 1995). Protein concentrations were determined from the previously reported extinction coefficients for each protein (Husain & Davidson, 1985; 1986; Husain et al., 1987). Nonlinear curve fitting of data was performed using either OLIS software or Sigma Plot 5.0 (Jandel Scientific). |
Falzon, L.,
Davidson, V. L. |
Kinetic Model for the Regulation by Substrate of Intramolecular Electron Transfer in Trimethylamine Dehydrogenase
Biochemistry; 1996; 35(7); 2445-2452. (Article) |
Experimental Procedures
Stopped-flow experiments were performed using an On-Line Instrument Systems (OLIS, Bogart, GA) RSM 1000 stopped-flow spectrophotometer. Data were collected and analyzed using OLIS software on an IBM compatible 486 personal computer. We have observed that the reduced forms of TMADH are quite stable against reoxidation by air, and therefore, anaerobic conditions were not necessary for these experiments. TMADH (2-4 µM) was mixed with various concentrations of trimethylamine (50 µM to 4 mM) in 0.1 M potassium phosphate buffer (pH 7.5) at 30 °C. The rates of formation of FMNH2 (represented by the change in absorbance at 443 nm) and the subsequent one-electron oxidation to form the flavinsemiquinone (represented by the change in absorbance at 365 nm) were slow enough under these conditions to be measured by stopped-flow spectroscopy (Figure 1). |
Chowdhury, S.,
Banerjee, R. |
Role of the Dimethylbenzimidazole Tail in the Reaction Catalyzed by Coenzyme B12-Dependent Methylmalonyl-CoA Mutase
Biochemistry; 1999; 38(46); 15287-15294. |
Methods
Equilibrium Binding Constants Measured by UV-Visible Absorption Spectroscopy. Binding of AdoCbl and AdoCbi to methylmalonyl-CoA mutase was followed spectrophotometrically using a Cary-118 spectrophotometer (Olis Instruments), in which the cuvette holder was maintained at 4 °C by a thermostated water circulator.
Stopped-Flow Fluorescence Spectroscopy. Rapid-scanning stopped-flow kinetic measurements were made using the RSM-2 software for the OLIS stopped-flow fluorescence spectrophotometer. An external water bath was used to maintain the temperature of the loading syringes at 30 °C. The concentration of AdoCbl was at least 8-fold greater than the enzyme concentration to obtain pseudo-first-order conditions. Fluorescence emission spectra were recorded between 310 and 450 nm using an emission slit width of 3.16 µm acquired at 62 scans/s or 31 scans/s depending on the time course of the decay. The excitation wavelength was 282 nm, and two consecutive slits of 0.6 µm width were used. |
Seravalli, J.,
Shoemaker, R. K.,
Sudbeck, M. J.,
Ragsdale, S. W. |
Binding of (6R,S)-Methyltetrahydrofolate to Methyltransferase from Clostridium thermoaceticum: Role of Protonation of Methyltetrahydrofolate in the Mechanism of Methyl Transfer
Biochemistry; 1999; 38(18); 5736-5745. |
Materials and Methods
Proton Uptake Experiments. Proton uptake titrations were performed on a modified Cary-14 spectrophotometer from OLIS (On-Line Instrument Systems Inc., Bogart, GA) at 25 °C. The temperature of the reaction was controlled by a circulating water bath. |
Seravalli, J.,
Zhao, S.,
Ragsdale, S. W. |
Mechanism of Transfer of the Methyl Group from (6S)-Methyltetrahydrofolate to the Corrinoid/Iron-Sulfur Protein Catalyzed by the Methyltransferase from Clostridium thermoaceticum: A Key Step in the Wood-Ljungdahl Pathway of Acetyl-CoA Synthesis
Biochemistry; 1999; 38(18); 5728-5735. |
Materials and Methods
Determination of kcat and kcat/Km for the CFeSP. Steady-state experiments were performed on a modified Cary-14 spectrophotometer (On-Line Instrument Systems Inc.) at 25 °C. The temperature of the reaction was controlled by a circulating water bath. For initial rate studies, the concentration of (6RS)-CH3-H4folate was fixed at 58 M and the CFeSP concentration was varied. |
Simianu, M.,
Murakami, E.,
Brewer, J. M.,
Ragsdale, S. W. |
Purification and Properties of the Heme- and Iron-Sulfur-Containing Heterodisulfide Reductase from Methanosarcina thermophila
Biochemistry; 1998; 37(28); 10027-10039. (Article) |
Materials and Methods
Spectroscopic and Spectroelectrochemical Methods. Electronic absorption spectra were acquired with a Beckman DU Series 7000 Diode Array Spectrophotometer or a Cary-14 Spectrophotometer modified by Olis (Bogart, GA). For low-temperature electronic absorption at 77 K, a dual-beam Aminco DW-2000TM UV-Visible Spectrophotometer equipped with a liquid nitrogen cooled cell was used.
Figure 3. Room temperature spectra of purified HDR. Spectra were recorded with a Cary 14 spectrophotometer (modified by OLIS, Inc., Bogart, GA) using a 1-cm pathway cuvette under an N2 atmosphere. The enzyme concentration was 2 µM (αβ dimeric protein). (A) Solid line, HDR as-isolated; dotted line, oxidized by opening the solution to air. (B) Solid line, reduced by treating with a 50-fold excess of sodium dithionite; dotted line, CO adduct obtained by exposing a solution of reduced HDR to 1 atm of CO. (C) Solid line, difference spectra of dithionite-reduced minus oxidized; dotted line, difference spectrum of CO add |
Menon, S.,
Ragsdale, S. W. |
Role of the [4Fe-4S] Cluster in Reductive Activation of the Cobalt Center of the Corrinoid Iron-Sulfur Protein from Clostridium thermoaceticum during Acetate Biosynthesis
Biochemistry; 1998; 37(16); 5689-5698. (Article) |
Materials and Methods
EPR and Circular Dichroism (CD) Spectroscopy and Spectroelectrochemistry.
CD measurements were made at 25 °C on a Olis-RSM CD system equipped with a DeSa monochromator and a Xenon Arc lamp. |
Seravalli, J.,
Kumar, M.,
Lu, W.P.,
Ragsdale, S. W. |
Mechanism of Carbon Monoxide Oxidation by the Carbon Monoxide Dehydrogenase/Acetyl-CoA Synthase from Clostridium thermoaceticum: Kinetic Characterization of the Intermediates
Biochemistry; 1997; 36(37); 11241-11251. (Article) |
Materials and Methods
Reductive CODH/ACS Titrations with Dithionite and CO.
Optical titrations were carried out in a 3 mL Pyrex cuvette (Spectrocell, Skokie, IL) modified at the top to fit a 20 mm black butyl rubber anaerobic stopper. Additions were made using gastight Hamilton syringes. Titrations were performed at 25 °C in buffers containing 50 mM Tris, pH 7.60, with PCA and PCD. Optical titrations were monitored at 420 nm in a Cary-14 spectrophotometer modified by Olis (Bogart, GA). |
Neyhart, G. A.,
Hupp, J. T.,
Curtis, J. C.,
Timpson, C.J.,
Meyer, T. J. |
Solvent-Induced Electron Transfer and Delocalization in Mixed-Valence Complexes. Electrochemistry
Journal of the American Chemical Society; 1996; 118(15); 3724-3729. (Article) |
| Measurements. Variable temperature spectra were obtained by using an Oxford Instruments DN1704 liquid nitrogen dewar and No. 3120 temperature controller in the sample compartment of a Cary 14 spectrophotometer modified by On Line Instrument Systems (OLIS) of Bogart, Georgia. Spectra were acquired by using software developed by OLIS. The reference cell was at room temperature. Solvent versus solvent baselines were recorded at each temperature for baseline subtraction. |
Brasch, N. E.,
Buckingham, D. A.,
Evans, A. B.,
Clark, C. R. |
17O NMR Study of Chromium(VI) Ions in Water
Journal of the American Chemical Society; 1996; 118(34); 7969-7980. (Article) |
Experimental Section
Spectrophotometric Rate Measurements.
All data were collected using an OLIS 3820 stopped-flow data collection system in conjunction with a Northstar Horizon computer interfaced to the Durrum spectrophotometer. Data were treated using the OLIS nonlinear least-squares fitting routine Versatile Data Fit. Approach to equilibrium was first-order in all cases. For each experiment a separate solution was made up by mixing equal volumes of the two reagents and the pH recorded. |
