A Heme Propionate Staples the Structure of Cytochrome for Methionine Ligation to the Heme Iron.

Ligand-switch reactions at the heme iron are common in biological systems, but their mechanisms and the features of the polypeptide fold that support dual ligation are not well understood. In cytochrome (cyt ), two low-stability loops (Ω-loop C and Ω-loop D) are connected by the heme propionate HP6. At alkaline pH, the native Met80 ligand from Ω-loop D switches to a Lys residue from the same loop.

DEPC modification of the Cu protein from Thermus thermophilus.

The Cu center is the initial electron acceptor in cytochrome c oxidase, and it consists of two copper ions bridged by two cysteines and ligated by two histidines, a methionine, and a carbonyl in the peptide backbone of a nearby glutamine. The two ligating histidines are of particular interest as they may influence the electronic and redox properties of the metal center. To test for the presence of reactive ligating histidines, a portion of cytochrome c oxidase from the bacteria Thermus thermophilus that contains the Cu site (the TtCu protein) was treated with the chemical modifier diethyl pyrocarbonate (DEPC) and the reaction followed through UV-visible, circular dichroism, and electron paramagnetic resonance spectroscopies at pH 5.

The K79G Mutation Reshapes the Heme Crevice and Alters Redox Properties of Cytochrome c.

The two roles of cytochrome c (cyt c), in oxidative phosphorylation and apoptosis, critically depend on redox properties of its heme iron center. The K79G mutant has served as a parent protein for a series of mutants of yeast iso-1 cyt c. The mutation preserves the Met80 coordination to the heme iron, as found in WT* (K72A/C102S), and many spectroscopic properties of K79G and WT* are indistinguishable.

A Compact Structure of Cytochrome c Trapped in a Lysine-Ligated State: Loop Refolding and Functional Implications of a Conformational Switch.

It has been suggested that the alkaline form of cytochrome c (cyt c) regulates function of this protein as an electron carrier in oxidative phosphorylation and as a peroxidase that reacts with cardiolipin (CL) during apoptosis. In this form, Met80, the native ligand to the heme iron, is replaced by a Lys. While it has become clear that the structure of cyt c changes, the extent and sequence of conformational rearrangements associated with this ligand replacement remain a subject of debate.

Application of radical cation spin density maps toward the prediction of photochemical reactivity between N-methyl-1,2,4-triazoline-3,5-dione and substituted benzenes.

Visible light irradiation of N-methyl-1,2,4-triazoline-3,5-dione in the presence of substituted benzenes is capable of inducing substitution reactions where no reaction takes place thermally. In addition to the formation of 1-arylurazole products resulting from ring substitution, side-chain substitution occurs in some cases where benzylic hydrogens are accessible to form benzylic urazole products. Formation of both types of products is most consistent with the involvement of a common intermediate, a radical ion pair, generated from photoexcitation of an initially formed charge-transfer complex.

The solution electrochemistry of tetrahydrobiopterin revisited.

Re-investigation of the electrochemical behavior of the nitric oxide synthase (NOS) cofactor tetrahydrobiopterin on graphite electrodes has revealed drastic differences in reversibility of electron transfer (ET) depending on the type of electrode surface employed. In particular, slow electron transfer kinetics and quasireversibility on an unpolished glassy carbon electrode can mask underlying concerted two-electron transfer chemistry and cause the appearance of an apparent one-electron couple. Nonetheless, the thermodynamic instability of the radical intermediate prevents any detectable build-up of this intermediate under any conditions tested.

Solvent isotope effects on interfacial protein electron transfer in crystals and electrode films.

D(2)O-grown crystals of yeast zinc porphyrin substituted cytochrome c peroxidase (ZnCcP) in complex with yeast iso-1-cytochrome c (yCc) diffract to higher resolution (1.7 A) and pack differently than H(2)O-grown crystals (2.4-3.

Electrochemical studies of arsenite oxidase: an unusual example of a highly cooperative two-electron molybdenum center.

Arsenite oxidase from Alcaligenes faecalis, an unusual molybdoenzyme that does not exhibit a Mo(V) EPR signal during oxidative-reductive titrations, has been investigated by protein film voltammetry. A film of the enzyme on a pyrolytic graphite edge electrode produces a sharp two-electron signal associated with reversible reduction of the oxidized Mo(VI) molybdenum center to Mo(IV). That reduction or oxidation of the active site occurs without accumulation of Mo(V) is consistent with the failure to observe a Mo(V) EPR signal for the enzyme under a variety of conditions and is indicative of an obligate two-electron center.

Fast, long-range electron-transfer reactions of a "blue" copper protein coupled non-covalently to an electrode through a stilbenyl thiolate monolayer.

A self-assembled monolayer (SAM), formed by the insitu saponification of a stilbenyl thioacetate on a gold electrode, yields fast electron transfer (ET)(the exchange rate at zero driving force exceeds 1600 s-1) with adsorbed molecules of the blue copper protein, azurin, over a distance exceeding 15 angstroms .

Voltammetric studies of the catalytic mechanism of the respiratory nitrate reductase from Escherichia coli: how nitrate reduction and inhibition depend on the oxidation state of the active site.

The respiratory molybdoenzyme nitrate reductase (NarGHI) from Escherichia coli has been studied by protein film voltammetry, with the enzyme adsorbed on a rotating disk pyrolytic graphite edge (PGE) electrode. Catalytic voltammograms for nitrate reduction show a complex wave consisting of two components that vary with pH, nitrate concentration, and the presence of inhibitors. At micromolar levels of nitrate, the activity reaches a maximum value at approximately -25 mV and then decreases as the potential becomes more negative.

Enzyme electrokinetics: using protein film voltammetry to investigate redox enzymes and their mechanisms.

Protein film voltammetry is a relatively new approach to studying redox enzymes, the concept being that a sample of a redox protein is configured as a film on an electrode and probed by a variety of electrochemical techniques. The enzyme molecules are bound at the electrode surface in such a way that there is fast electron transfer and complete retention of the chemistry of the active site that is observed in more conventional experiments. Modulations of the electrode potential or catalytic turnover result in the movement of electrons to, from, and within the enzyme; this is detected as a current that varies in characteristic ways with time and potential.