Observation of an unprecedented Cu Bis-His site: crystal structure of the H129V mutant of nitrite reductase

Copper nitrite reductases contain both an electron-transfer type 1 Cu site and a catalytic type 2 Cu site. We have mutated one of the type 2 copper ligating histidines to observe the effect on catalytic turnover. This mutation has created a unique site where Cu is ligated by 2 His Nepsilon2 atoms alone.     

Products from the reaction of M3(CO)12 (M = Ru or Os) with water

Reaction of the carbonyl Ru₃(CO)₁₂ with water leads to the formation of polynuclear hydrides α-H₄Ru₄(CO)₁₂, α-H₂Ru₄(CO)₁₃; the corresponding reaction with Os₃(CO)₁₂ yields the complexes (H)(OH)Os₃(CO)₁₀, H₂Os₄(CO)₁₃, H₄Os₄(CO)₁₂, H₂Os₅(CO)₁₆, H₂Os₅(CO)₁₅, H₂Os₆(CO)₁₈ and H₂Os₇(CO)₁₉C.     

N‐Butylpyridinium undecachlorocarbadodecaborate and comparison with similar compounds

The title compound, C₉H₁₄N⁺·CHB₁₁Cl₁₁⁻, was obtained in the course of our continuing studies of the low‐melting salts of closo‐ and nido‐carborane cage anions with alkylpyridinium and dialkylimidazolium cations. The title compound is the first example of a pyridinium salt of a perchlorinated carborane anion. The structure consists of one N‐butylpyridinium cation counterbalanced by one perchlorinated carborane cage anion per asymmetric unit. By changing the counter‐ion, different packings are observed, and to try to understand this the new structure is compared with five similar compounds.     

Speech intonation and focus location in matched statements and questions

An acoustical study of speech production was conducted to determine the manner in which the location of linguistic focus influences intonational attributes of duration and fundamental voice frequency (F0) in matched statements and questions. Speakers orally read sentences that were preceded by aurally presented stimuli designed to elicit either no focus or focus on the first or last noun phrase of the target sentences. Computer-aided acoustical analysis of word durations showed a localized, large magnitude increase in the duration of the focused word for both statements and questions. Analysis of F0 revealed a more complex pattern of results, with the shape of the F0 topline dependent on sentence type and focus location. For sentences with neutral or sentence-final focus, the difference in the F0 topline between questions and statements was evident only on the last key word, where the F0 peak of questions was considerably higher than that of statements. For sentences with focus on the first key word, there was no difference in peak F0 on the focused item itself, but the F0 toplines of questions and statements diverged quite dramatically following the initial word. The statement contour dropped to a low F0 value for the remainder of the sentence, whereas the question remained quite high in F0 for all subsequent words. In addition, the F0 contour on the focused word was rising in questions and falling in statements, regardless of focus location. The results provide a basis for work on the perception of linguistic focus.     

A distal pocket Leu residue inhibits the binding of O2 and NO at the distal heme site of cytochrome c'

Cytochromes c' are pentacoordinate heme proteins with sterically hindered distal sites that bind NO and CO but do not form stable complexes with O(2). Removal of distal pocket steric hindrance via a Leu→Ala mutation yields favorable O(2) binding (K(d) ~49 nM) without apparent H-bond stabilization of the Fe-O(2) moiety, as well as an extremely high distal heme-NO affinity (K(d) ~70 fM). The native Leu residue inhibits distal coordination of diatomic ligands by decreasing k(on) as well as increasing k(off). The connection between distal steric constraints, k(off) values, and distal to proximal heme-NO conversion is discussed.     

Probing the function of Mycobacterium tuberculosis catalase-peroxidase by site-directed mutagenesis

Catalase-peroxidase is a multi-functional heme-dependent enzyme which is well known for its ability to carry out both catalatic and peroxidatic reactions. Catalase-peroxidase from Mycobacterium tuberculosis(mtCP) is of particular interest because this enzyme activates the pro-antitubercular drug isoniazid. It is estimated that 2 billion people are infected with M. tuberculosis, the principal causative agent of tuberculosis, and that 2 million people die from the disease each year. The rise of drug-resistant strains continues to be of critical concern and it is well documented that mutations which reduce activity or inactivate mtCP lead to increased levels of isoniazid resistance in M. tuberculosis. The recent determination of the crystal structure for M. tuberculosis mtCP has aided the understanding of how the enzyme functions and provides a three-dimensional framework for testing hypotheses about the roles of various residues in the active site. Here we report site-directed mutagenesis studies of three conserved residues located near the heme of mtCP, His-108, Trp-107 and Trp-321 including the construction of the double mutant W107F-W321F. Resulting mutants have been purified and their catalatic and peroxidatic activities have been determined. Data are compared in the context of related studies aimed at dissecting the roles of these residues in the different activities of the enzyme. Analyses of single and double mutants studied here emphasise that the hydrogen bonding network surrounding the heme in the active site appears more important for maintenance of catalatic rather than peroxidatic activity in CP enzymes.     

Active site structures and the redox properties of blue copper proteins: atomic resolution structure of azurin II and electronic structure calculations of azurin, plastocyanin and stellacyanin

Understanding how the active site structures of blue copper proteins determine their redox properties is the central structure-function relationship question of this important class of protein, also referred to as cupredoxins. We here describe both experimental and computational studies of azurin, plastocyanin and stellacyanin designed to define more accurately the geometric structures of the active site of the reduced and oxidized species, and thus to understand how these structures determine the redox potentials of these proteins. To this end the crystal structure of reduced azurin II has been determined at an atomic resolution of 1.13 Angstrom and is presented here. Co-ordinates and structure factors have been deposited in the RCSB Protein Data Bank with accession codes 2ccw and r2ccwsf respectively. The improved accuracy provided by the atomic resolution for the metal stereochemistry are utilised in conjunction with the EXAFS data for theoretical calculations. Multilevel calculations involving density functional theory and molecular mechanical potentials are used to predict both the geometric and electronic structure of the active sites of azurin, plastocyanin and stellacyanin and to estimate the relative redox potentials of these three proteins. We have also compared the relative energies of the structures obtained from experiment at varying resolutions, and from the isolated and embedded cluster calculations. We find significant energy differences between low and high (atomic) resolution structures arising primarily due to inaccuracies in the Cu-ligand distances in the lower resolution structures, emphasising the importance of accurate, very high resolution structural information. QM/MM structures are only approximately 1 kcal mol(-1) lower in energy than the 1.13 Angstrom structure while the optimized gas phase structure is 13.0 kcal mol(-1) lower in energy.