T. M. Duncan

Infrared spectra of chemisorbed CO on Rh

The infrared spectrum of CO chemisorbed on alumina‐supported Rh atoms has been investigated. In agreement with previous work, three types of adsorbed species have been clearly distinguished on the basis of their C–O stretching frequencies. Species I, assigned as Rh(CO)2, is formed only with Rh atoms which are isolated from each other. Species II, assigned as Rh–CO, and III, assigned as Rh2CO, are formed on Rh clusters having two or more Rh atoms. CO‐species II and III undergo interactions with neighbor CO species causing an increase in wave number as coverage increases. Based on infrared intensity measurements for species I, the OC–Rh–CO angle is ∼90°. Chemisorbed 13CO yields the expected infrared spectrum on Rh, and rapid isotopic exchange between 13CO(ads) and 12CO(g) is observed which cannot be explained by the observed rate of desorption of CO from the supported Rh surface.

Structure of the ATP synthase catalytic complex (F(1)) from Escherichia coli in an autoinhibited conformation.

ATP synthase is a membrane-bound rotary motor enzyme that is critical for cellular energy metabolism in all kingdoms of life. Despite conservation of its basic structure and function, autoinhibition by one of its rotary stalk subunits occurs in bacteria and chloroplasts but not in mitochondria. The crystal structure of the ATP synthase catalytic complex (F1) from Escherichia coli described here reveals the structural basis for this inhibition. The C-terminal domain of subunit ɛ adopts a heretofore unknown, highly extended conformation that inserts deeply into the central cavity of the enzyme and engages both rotor and stator subunits in extensive contacts that are incompatible with functional rotation. As a result, the three catalytic subunits are stabilized in a set of conformations and rotational positions distinct from previous F1 structures.

Exploiting the Nitrilotriacetic Acid Moiety for Biolabeling with Ultrastable Perylene Dyes

Fluorescent probes are essential for the exploration of protein function, detection of molecular interactions, and conformational changes. The nitrilotriacetic acid derivatives of different chromophores were successfully used for site-selective noncovalent fluorescence labeling of histidine-tagged proteins. All of them, however, suffer from the same drawback--loss of the fluorescence upon binding of the nickel ions. Herein we present the solution and solid phase synthesis of water-soluble perylene(dicarboximide) functionalized with a nitrilotriacetic acid moiety (PDI-NTA). The photophysical properties of PDI-NTA revealed an exceptional photostability and fluorescence quantum yield that remained unchanged upon addition of nickel ions. The F1 complex of F0F1-ATP synthase from Escherichia coli, containing three hexahistidine tags, was labeled and the suitability for site-specific labeling of the new chromophore demonstrated using fluorescence correlation spectroscopy.

Chemisorption and surfaces studied by nuclear magnetic resonance spectroscopy

Abstract This paper presents an introduction to the study of surfaces and chemically adsorbed species with nuclear magnetic resonance (NMR) spectroscopy. The analysis is based on nuclear magnetic interactions in the solid state: dipole-dipole couplings, chemical shift anisotropy, Knight shifts, and quadrupolar splitting. The physical origins and characteristics of each interaction, as well as relative intensities for different nuclei, are discussed. In particular, emphasis is placed on the relation of these interactions to quantities of interest to studies in adsorption and catalysis: motional properties of the adsorbate, the distribution of adsorption sites, the chemical state of atoms adsorbed at the surface, electrostatic field gradients, and the metallic character of surface atoms. Techniques to observe these interactions are described; subdivided by the type of nucleus: strongly coupled nuclei (e.g. 1H, 19F), weakly coupled nuclei (e.g. 13C, 15N, 29Si, 195Pt), and quadrupolar nuclei (e.g. 2H, 14N, 27Al). The techniques described to isolate and identify the overlapping effects in the spectra include multiple-pulse spin echoing and decoupling, double-resonance irradiation, multiple-quantum excitation, and mechanical sample spinning. A review of the recent application of these techniques to studies of adsorption and surfaces illustrates the potentials and limitations. Finally, a procedure for formulating a NMR study of surface samples is proposed, with respect to sample composition and character, and the type of information desired.

Subunit rotation in F0F1-ATP synthases as a means of coupling proton transport through F0 to the binding changes in F1

The rotation of an asymmetric core of subunits in F0F1-ATP synthases has been proposed as a means of coupling the exergonic transport of protons through F0 to the endergonic conformational changes in F1 required for substrate binding and product release. Here we review earlier evidence both for and against subunit rotation and then discuss our most recent studies using reversible intersubunit disulfide cross-links to test for rotation. We conclude that the γ subunit of F1 rotates relative to the surrounding catalytic subunits during catalytic turnover by both soluble F1 and membrane-bound F0F1. Furthermore, the inhibition of this rotation by the modification of F0 with DCCD suggests that rotation in F1 is obligatorily coupled to rotation in F0 as an integral part of the coupling mechanism.

A 13C NMR study of the adsorbed states of CO on Rh dispersed on Al2O3

The results of nuclear magnetic resonance (NMR) spectroscopy have been analyzed with respect to previous infrared studies of CO adsorbed on Rh dispersed on Al2O3 to quantify the site distribution and to describe the adsorbed state. The 13C NMR spectra account for all the 13CO adsorbed on a 2.2% Rh on Al2O3 substrate. Although the spectra from the different adsorbed states of CO overlap, the line shapes may be separated into two components based on differences in the 13C spin–lattice relaxation times. These two components have been assigned to the 13CO dicarbonyl formed on single Rh atoms and to 13CO adsorbed on Rh rafts. The component attributed to the CO adsorbed on the raft sites is further separated into linear and bridged CO state contributions based on chemical shift information, yielding a quantitative distribution of the three adsorbed states of CO on Rh. The 13CO distribution is used to estimate the molar integrated intensities of the infrared spectrum of 13CO on Rh at high coverage and to determi...

Study of fluorine in silicate glass with 19F nuclear magnetic resonance spectroscopy

We report an application of nuclear magnetic resonance (NMR) spectroscopy to the study of fluorine‐doped silicate glass prepared by the modified chemical vapor deposition process, prior to drawing the rod into fibers. The silica contains 1.03‐wt.u2009% fluorine, as determined by the calibrated intensity of the 19F NMR spectrum. The isotropic chemical shift of the 19F spectrum shows that fluorine bonds only to silicon; there is no evidence of oxyfluorides. Analysis of the distribution of nuclear dipolar couplings between fluorine nuclei reveals that the relative populations of silicon monofluoride sites [Si(O–)3F] and species having near‐neighbor fluorines, such as silicon difluoride sites [Si(O–)2F2], are nearly statistically random. That is, to a good approximation, the fluorine substitutes randomly into the oxygen sites of the silica network. There is no evidence of local clusters of fluorine sites, silicon trifluoride sites [Si(O–)F3], or silicon tetrafluoride (SiF4).

Energy-driven subunit rotation at the interface between subunit a and the c oligomer in the FO sector of Escherichia coli ATP synthase

Subunit rotation within the F1 catalytic sector of the ATP synthase has been well documented, identifying the synthase as the smallest known rotary motor. In the membrane-embedded FO sector, it is thought that proton transport occurs at a rotor/stator interface between the oligomeric ring of c subunits (rotor) and the single-copy a subunit (stator). Here we report evidence for an energy-dependent rotation at this interface. FOF1 was expressed with a pair of substituted cysteines positioned to allow an intersubunit disulfide crosslink between subunit a and a c subunit [aN214C/cM65C; Jiang, W. & Fillingame, R. H. (1998) Proc. Natl. Acad. Sci. USA 95, 6607–6612]. Membranes were treated with N,N′-dicyclohexyl-[14C]carbodiimide to radiolabel the D61 residue on less than 20% of the c subunits. After oxidation to form an a–c crosslink, the c subunit properly aligned to crosslink to subunit a was found to contain very little 14C label relative to other members of the c ring. However, exposure to MgATP before oxidation significantly increased the radiolabel in the a–c crosslink, indicating that a different c subunit was now aligned with subunit a. This increase was not induced by exposure to MgADP/Pi. Furthermore, preincubation with MgADP and azide to inhibit F1 or with high concentrations of N,N′-dicyclohexylcarbodiimide to label most c subunits prevented the ATP effect. These results provide evidence for an energy-dependent rotation of the c ring relative to subunit a.

The characterization of carbonaceous species on ruthenium catalysts with 13C nuclear magnetic resonance spectroscopy

13C Nuclear magnetic resonance (NMR) spectroscopy has been applied to the study of carbon species deposited on supported and unsupported ruthenium catalysts during CO hydrogenation. Four forms of nonoxygenated carbon on the substrate have been identified, designated Cα, Cβ1, Cβ2, and unreactive carbon. Correlation of isotropic shifts, nuclear dipolar interactions, and anisotropy of chemical shielding leads to a description of each carbon species. Cα is interpreted as carbidic carbon atoms distributed in a variety of sites located on or below the metal surface. Cβ1and Cβ2 are alkyl groups attached to the catalyst and are differentiated by their relative mobilities and interconversion to other forms. Cβ1, reorients at room temperature but the motion is quenched at 100 K. Cβ2, is motionally averaged at 110 K and is selectively depleted by purging the catalyst with an inert gas. The unreactive carbon has a 13C NMR spectrum similar to that of turbostratic graphite. The relationships of the different carbon species to each other are discussed.

ATP hydrolysis by membrane-bound Escherichia coli F0F1 causes rotation of the γ subunit relative to the β subunits

We recently demonstrated that the gamma subunit in soluble F1-ATPase from Escherichia coli rotates relative to surrounding beta subunits during catalytic turnover (Duncan et al. (1995) Proc. Natl. Acad. Sci. USA 92, 10964-10968). Here, we extend our studies to the more physiologically relevant membrane-bound F0F1 complex. It is shown that beta D380C-F1, containing a beta-gamma intersubunit disulfide bond, can bind to F1-depleted membranes and can restore coupled membrane activities upon reduction of the disulfide. Using a dissociation/reconstitution approach with crosslinked beta D380C-F1, beta subunits containing an N-terminal Flag epitope (beta flag) were incorporated into the two non-crosslinked beta positions and the hybrid F1 was reconstituted with membrane-bound F0. Following reduction and ATP hydrolysis, reoxidation resulted in a significant amount of crosslinking of beta flag to the gamma subunit. This demonstrates that gamma rotates within F1 during catalytic turnover by membrane-bound F0-F1. Furthermore, the rotation of gamma is functionally coupled to F0, since preincubation with DCCD to modify F0 blocked rotation.