Exogenous Ligand Binding Research Articles

Accessibility to the active site of methane monooxygenase: the first demonstration of exogenous ligand binding to the diiron cluster

Accessibility to the active site of methane monooxygenase: the first demonstration of exogenous ligand binding to the diiron cluster

Structural and spectroscopic characterization of exogenous ligand binding to isolated factor F430 and its configurational isomers

Binding of axial ligands to the nickel(II) of isolated factor F430 from the methyl reductase enzyme of Methanobacterium thermoautotrophicum is demonstrated. Evidence of bis-ligand coordination is obtained from the x-ray absorption, optical, and resonance Raman spectral characterization of F430 and its 12,13-diepimeric isomer in the presence of a large excess of cyanide, pyridine, or 1-methylimidazole. Significant broadening and 5-10-nm red shifts of the main 430-nm optical absorption band and shifts of up to 30 cm-1 for the high-frequency Raman lines are observed upon coordination of these axial ligands. The Raman spectra of native F430 and the diepimer with a particular axial ligand are nearly identical. Nickel x-ray absorption edge spectra of the diepimer in the absence and presence of these exogenous ligands are indicative of conversion from a square-planar to a tetragonally distorted octahedral geometry. Analyses of the nickel extended x-ray absorption fine structure data for the ligated diepimer complexes yield detailed structural information for these complexes. Implications of these data with respect to the enzymatic mechanism and the structure of the enzyme-bound factor are discussed.

Ligand binding to cytochrome c and other related haem proteins and peptides. Part I. Equilibrium studies

Investigation of ligand binding to native cytochrome c, carboxymethyl-Met 80-cytochrome c, myoglobin and haemhexapeptide revealed that the binding of exogenous ligands is modulated by the following factors: 1. Hydrophobicity of the haem environment. 2. Haem accessibility to exogenous ligands, termed the haem crevice ‘open-closed’ parameter. 3. Steric interactions between the protein and the bound ligand.

17O]Water and nitric oxide binding by protocatechuate 4,5-dioxygenase and catechol 2,3-dioxygenase. Evidence for binding of exogenous ligands to the active site Fe2+ of extradiol dioxygenases.

Pseudomonas testosteroni protocatechuate 4,5-dioxygenase and Pseudomonas putida catechol 2,3-dioxygenase (metapyrocatechase) catalyze extradiol-type oxygenolytic cleavage of the aromatic ring of their substrates. The essential active site Fe2+ of each enzyme binds nitric oxide (NO) to produce an EPR active complex with an electronic spin of S = 3/2. Hyperfine broadening of the EPR resonances of the nitrosyl complexes by 17O-enriched H2O shows that water is bound directly to the Fe2+ in the native enzymes, but is apparently displaced in substrate complexes. NO is not displaced by either substrates or inhibitors. The EPR spectra of several enzyme-inhibitor-NO complexes are different from those of enzyme-NO or enzyme-substrate-NO complexes and are found to be broadened by 17O-enriched water. The data show that at least 2 and perhaps 3 sites in the Fe ligation can be occupied by exogenous ligands. Furthermore, it is likely that substrates and inhibitors displace water by binding either at or near to the Fe in the nitrosyl complex. Nitric oxide binding is found to be substrate-dependent for each enzyme. Native catechol 2,3-dioxygenase exhibits KD values of 190 microM and 2.0 mM for NO binding in two types of independent sites. Only one type of site is observed in the catechol complex which exhibits a KD for NO of 3.4 microM. One type of NO binding site is observed for both the native and substrate complexed protocatechuate 4,5-dioxygenase with KD values of 360 and 3 microM, respectively. The presence of a specific site in the Fe coordination for NO which is modified in the substrate complex, suggests that O2 binding by the extradiol dioxygenases may also occur at the Fe.

Function and regulation of hepatic and intestinal fatty acid binding proteins

Two structurally different fatty acid binding proteins (FABP) have been isolated from rat liver and small intestinal epithelium. hFABP is a 14 184 Da protein found in abundance in both liver and small intestine, whereas gFABP (15 063 Da) is abundantly present only in small intestine. This review discusses studies which have provided insight into the physiological functions of these proteins. These include analyses of endogenous and exogenous ligand binding to FABP in vitro; examination of the modulating effect of FABP preparations on enzyme activities in vitro; exploration of relationships between alterations in cytosolic FABP content in response to hormonal, pharmacological, and dietary manipulations and changes in the rates of cellular fatty acid uptake and utilization; and studies of hFABP turnover and the mechanisms of FABP regulation. These experiments provide compelling evidence for a broad role of the FABPs in the transport, utilization and cellular economy of free fatty acids in the liver and small intestine, and also in protecting several aspects of cellular function against the modulatory effects of fatty acids, fatty acyl-CoA esters, and other ligands. Studies of FABP regulation also suggest a role in long-term rather than short-term modulation .of hepatic fatty acid metabolism and indicate that hFABP and gFABP may perform different functions in the small intestine.

Model complexes for the active sites of reduced and oxidized sites of hemocyanin and tyrosinase. Structures of binuclear Cu(I) and Cu(II) complexes and characterization of a model copper monooxygenase reaction

It has been established that the dioxygen carrying copper containing protein hemocyanin and the copper monooxygenase tyrosinase both contain electronically coupled binuclear copper centers. In the deoxy protein, copper centers are thought to be two or three coordinate, while in their oxidized form, tetragonally coordinated Cu(II) ions are bridged by an endogenous protein group which is thought to be phenolate from tyrosine. Coordinated dioxygen (as peroxide) is also known to bridge the two coppers. The binding of other exogenous ligands (azide, chloride, etc.) to the binuclear copper centers has been used to probe the active site structures and some of these may also bridge the Cu(II) ions in methemocyanin or tyrosinase derivatives [1]. Extensive coordination model studies are being carried out [2–4] in order to 1) determine the nature of the ligand donors and coordination geometries of these active site copper centers 2) mimick and understand the coordination chemistry of relevant Cu(I) systems; 3) mimick the Cu(II) binuclear centers in order to relate the spectroscopic and chemical properties of these models and their derivatives (with appropriate probes) to those of the proteins and 4) understand those factors necessary and the mechanism(s) for reversible dioxygen binding and ‘activation’ of dioxygen ( i.e. specific hydroxylation of substrates by the copper monooxygenases). As part of our own extensive investigations into the chemistry of binuclear Cu(I) and Cu(II) moieties, we have employed the binucleating ligand m-XYLpy, I (py = 2-pyridyl), where two tridentate donor groups are separated by a meta-xylyl bridge [5]. A novel binuclear cuprous complex, II, ( See next column) containing well separated trigonally coordinated Cu(I) ions is isolated by the reaction of I with two equivalents of Cu(CH 3CN) 4PF 6 [6]. The coordination geometry and the type of ligands in II makes it a good model complex for the deoxy-sites in the proteins. In addition, II reacts with O 2 in a manner analogous to the copper monooxygenase tyrosinase. Specific hydroxylation of the xylyl ligand occurs producing the phenoxy- and hydroxy- (R = H) doubly bridged binuclear Cu(II) complex III. Manometric O 2 uptake experiments with II and mass spectrometric analyses of the oxidation product III utilizing isotopically labeled dioxygen demonstrate that both atoms of oxygen in III are derived from 18O 2[6, 7]: ▪ The structures of compounds II and III have been proven by X-ray crystallographic studies [5–7]. Compounds III contain tetragonally coordinated Cu(II) ions which are phenolate bridged (Cu⋯Cu = ∼3.1 Å), making them excellent structural models for the proposed active sites in hemocyanin and tyrosinase. Reaction of III (R = H) with HX (X = N 3 − , Cl − results in the replacement of the bridging ligand (-OH) by X with the formation of complexes IV. These have also been structurally characterized and they exhibit coordination geometries very similar to III. In the case of the azide complex (X = N 3 − ), a novel μ-1,1-azido type of bridging is observed [7]. This mode of coordination has not been previously proposed to take place in the protein copper centers. Its possible occurrence should be considered in studies where azide ligand binding is used as a spectral probe of the protein copper centers.

Preparation and characterization of a stable half met derivative of type 2 depleted [formula omitted] laccase: Exogenous ligand binding to the type 3 site

We report the preparation and characterization of a stable half met (Cu(II)Cu(I)) type 2 copper depleted derivative of Rhus laccase. Anion binding studies to this mixed valent type 3 protein form indicate no tight binding of anions nor group 1 - group 2 ligand behavior. This suggests that, in contrast to the well-characterized hemocyanins and tyrosinase coupled binuclear sites, exogenous ligands do not appear to bridge the type 3 binuclear copper ions in laccase.

Anion binding to oxidized type 2 depleted and native laccase: A spectroscopically effective model for exogenous ligand binding to the type 3 - type 2 active site

Chemical and spectroscopic comparison of anion binding to the oxidized type 3 sites in type 2 depleted and native Rhus vernicifera laccase demonstrates that the type 3 - type 2 active site of laccase has an especially high affinity for exogenous ligands. N 3 − , O 2 2− and F − binding and competition, and resonance Raman studies, indicate that this high affinity is not due to strong, equatorial type 2 ligation, but rather to a structurally non-specific role of the type 2 copper in stabilizing anion binding at the type 3 binuclear cupric site. These studies, combined with previous results on half met type 2 depleted laccase, generate a spectroscopically effective model for peroxide birding to the type 3 - type 2 active site.