Denis A. Proshlyakov

From water to oxygen and back again: mechanistic similarities in the enzymatic redox conversions between water and dioxygen.

We propose that the interconversions of water and oxygen are catalyzed by the transition metal ions of Photosystem II and cytochrome c oxidase in remarkably similar ways. Oxygen-oxygen bond formation and cleavage occurs between two oxygen atoms that are bound as terminal ligands to two redox-active metal ions. Hydrogen atom transfer to or from a tyrosine residue is an essential component of the processes in both enzymes.

Equilibrium and Kinetic Behavior of Fe(CN)63−/4− and Cytochrome c in Direct Electrochemistry Using a Film Electrode Thin-Layer Transmission Cell

We report on the design and performance of a thin-layer electrochemical cell optimized for use with optically transparent film electrodes in combination with UV/vis and IR transmission spectroscopic measurements. The cell allows for measurements under both aerobic and anaerobic conditions. The direct, unmediated electron transfer, as assessed by the current transient, and the corresponding optical response observed for the Fe(CN)(6)(3-/4-) couple were in good agreement with theoretical predictions for voltammetry and optical absorption by an analyte confined in a thin layer. Chronoamperometric and spectroscopic measurements of Fe(CN)(6)(3-/4-) on gold mesh electrode revealed fast kinetics strongly influenced by the electrolyte concentration. Maximal apparent rates exceeding 2 s(-1) in 1 M KCl were observed optically. The direct kinetic and thermodynamic behavior of cytochrome c was compared with several electrode materials using the cell. The results showed heme ligand-dependent changes in the protein-electrode interactions. Mid-UV/visible spectral changes upon redox transitions in native cytochrome c and its cyanide derivative, as well as dissociation of the ferrous cytochrome c-CN complex, are reported.

Spectroscopic analyses of 2-oxoglutarate-dependent oxygenases: TauD as a case study

A wide range of spectroscopic approaches have been used to interrogate the mononuclear iron metallocenter in 2-oxoglutarate (2OG)-dependent oxygenases. The results from these spectroscopic studies have provided valuable insights into the structural changes at the active site during substrate binding and catalysis, thus providing critical information that complements investigations of these enzymes by X-ray crystallography, biochemical, and computational approaches. This mini-review highlights taurine hydroxylase (taurine:2OG dioxygenase, TauD) as a case study to illustrate the wealth of knowledge that can be generated by applying a diverse array of spectroscopic investigations to a single enzyme. In particular, electronic absorption, circular dichroism, magnetic circular dichroism, conventional and pulse electron paramagnetic, Mössbauer, X-ray absorption, and resonance Raman methods have been exploited to uncover the properties of the metal site in TauD.

Apoprotein isolation and activation, and vibrational structure of the Helicobacter mustelae iron urease.

The micro aerophilic pathogen Helicobacter mustelae synthesizes an oxygen-labile, iron-containing urease (UreA2B2) in addition to its standard nickel-containing enzyme (UreAB). An apoprotein form of the iron urease was prepared from ureA2B2-expressing recombinant Escherichia coli cells that were grown in minimal medium. Temperature-dependent circular dichroism measurements of holoprotein and apoprotein demonstrate an enhancement of thermal stability associated with the UreA2B2 metallocenter. In parallel to the situation reported for nickel activation of the standard urease apoprotein, incubation of UreA2B2 apoprotein with ferrous ions and bicarbonate generated urease activity in a portion of the nascent active sites. In addition, ferrous ions were shown to be capable of reductively activating the oxidized metallocenter. Resonance Raman spectra of the inactive, aerobically-purified UreA2B2 holoprotein exhibit vibrations at 495cm(-1) and 784cm(-1), consistent with ν(s) and ν(as) modes of an Fe(III)OFe(III) center; these modes undergo downshifts upon binding of urea and were unaffected by changes in pH. The low-frequency mode also exhibits an isotopic shift from 497 to 476cm(-1) upon (16)O/(18)O bulk water isotope substitution. Expression of subunits of the conventional nickel-containing Klebsiella aerogenes urease in cells grown in rich medium without nickel resulted in iron incorporation into a portion of the protein. The inactive iron-loaded species exhibited a UV-visible spectrum similar to oxidized UreA2B2 and was capable of being reductively activated under anoxic conditions. Results from these studies more clearly define the formation and unique properties of the iron urease metallocenter.

Metal and substrate binding to an Fe(II) dioxygenase resolved by UV spectroscopy with global regression analysis

The addition of divalent metal ions or substrate taurine to TauD, an alpha-ketoglutarate-dependent dioxygenase, alters its UV absorption, as clearly observed by monitoring the proteins difference spectra. Binding of metal ions leads to a decrease in absorption at approximately 297 nm and modulation of other features. A separate signature with enhanced absorption at approximately 295 nm is identified for binding of taurine. These narrow ( approximately 700 cm(-1)) and intense ( approximately 0.5mM(-1) cm(-1)) spectral changes are attributed to ligand-induced protein conformational changes affecting the environment of aromatic residues. The changes in the UV difference spectra were exploited to assess directly the thermodynamics and kinetics of ligand interactions in wild-type TauD and selected variants. This approach holds promise as a new tool to probe ligand-induced conformational changes in a wide range of other proteins. Experimental and quantification approaches for a reliable analysis of protein absorption below 320 nm are presented.

Resonance Raman Studies of Compounds I and II of Arthromyces ramosus Peroxidase: Close Similarities in Their Raman Spectra But Distinct Oxygen Exchangeability of the Fe=O Heme

Simultaneous measurements of resonance Raman and absorption spectra were performed for intermediates generated upon addition of hydrogen peroxide to ferric Arthromyces ramosus peroxidase (ARP) using the microcirculating system constructed in this laboratory, which enables generation of desirable intermediates under steady-state conditions. Compound I of ARP generated at neutral pH was stable over tens of minutes in the absence of laser illumination with this circulation system, but was gradually degraded under laser illumination, giving rise to a new irreversible species with an iron-oxo heme. Such photosensitivity was not observed for compound II in the steady state at alkaline pH. Surprisingly, the Raman spectrum of compound I of ARP in the high-frequency region, where characteristic frequency shifts are expected upon oxidation of the macrocycle, was quite close to that of compound II, despite the fact that the reduced Soret absorption indicated the formation of a π-cation radical. The Fe=O stretching (νFe=O) frequency of compound I was observed at 781 cm−1 for the 16O derivative but appeared as a doublet at 744 and 731 cm−1 for the 18O derivative. The isotope sensitivity of the νFe=O mode of compound I was seen upon H216O/H218O solvent substitution but not upon H216O2/H218O2 peroxide substitution in H216O at neutral pH. This directly indicates the occurrence of an oxygen atom exchange between the oxo-heme and bulk water, providing the first example of such exchange in compound I of peroxidases. The oxygen exchange was abolished for compound II at alkaline pH, for which the νFe=o mode was seen at 787/749 cm−1 only upon H216O2/H218O2 peroxide substitution. The oxygen exchangeability seems to depend on protonation of a nearby residue with pKα ∼ 9 and to correlate with stability of compound I.

Ligand Dynamics in the Binuclear Site in Cytochrome Oxidase

The dioxygen-reduction mechanism in cytochrome oxidase relies on proton control of the electron-transfer events that drive the process. Recent work on proton delivery and efflux channels in the protein that are relevant to substrate reduction and proton pumping is considered, and the current status of this area is summarized. Carbon monoxide photodissociation and the ligand dynamics that occur subsequent to photolysis have been valuable tools in probing possible coupling schemes for linking exergonic electron-transfer chemistry to endergonic proton translocation. Our picosecond-time-resolved Raman results show that the heme a 3-proximal histidine bond remains intact following CO photodissociation but that the local environment around the heme a 3 center in the photoproduct is in a nonequilibrium state. This photoproduct relaxes to its equilibrium configuration on the same time scale as ligand release occurs from CuB, which suggests a coupling between the two events and a potential signaling pathway between the site of O2 binding and reduction and the putative element, CuB, that links the redox chemistry to the proton pump.