Michelle A. Pressler

Spectroscopic characterization of metal binding by Klebsiella aerogenes UreE urease accessory protein

Abstract The urease accessory protein encoded by ureE from Klebsiella aerogenes is proposed to function in Ni(II) delivery to the urease apoprotein. Wild-type UreE contains a histidine-rich region at its carboxyl terminus and binds 5–6 Ni per dimer, whereas the functionally active but truncated H144*UreE lacks the histidine-rich motif and binds only two Ni per dimer [Brayman TG, Hausinger RP (1996) J Bacteriol 178 : 5410-5416]. For both proteins, Cu(II), Co(II), and Zn(II) ions compete for the Ni-binding sites. In order to characterize the coordination environments of bound metals, especially features that are unique to Ni, the Ni-, Cu-, and Co-bound forms of H144*UreE were studied by a combination of EPR, ESEEM, hyperfine-shifted 1H-NMR, XAS, and RR spectroscopic methods. For each metal ion, the two binding sites per homodimer were spectroscopically distinguishable. For example, the two Ni-binding sites each have pseudo-octahedral geometry in an N/O coordination environment, but differ in their number of histidine donors. The two Cu-binding sites have tetragonal geometry with two histidine donors each; however, the second Cu ion is bound by at least one cysteine donor in addition to the N/O-type donors found for the first Cu ion. Two Co ions are bound to H144*UreE in pseudo-octahedral geometry with N/O coordination, but the sites differ in the number of histidine donors that can be observed by NMR. The differences in coordination for each type of metal ion are relevant to the proposed function of UreE to selectively facilitate Ni insertion into urease in vivo.

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.

Dioxygen activation in enzymic systems and in inorganic models

Abstract Cytochrome oxidase reduces O 2 to water quickly and with low overpotential. In addition, it uses the exergonicity of this reaction to pump protons against their thermodynamic gradient, thus contributing directly to the chemiosmotic potential that is used to synthesize ATP. In recent work, we have developed means by which to use time-resolved resonance Raman to study transient heme iron-bound oxygen intermediates that occur during the reduction of O 2 by cytochrome oxidase. Thus far, five different oxygen isotope sensitive modes have been observed; the temporal behavior of each during the reaction sequence has been characterized roughly. By combining the structure-specific vibrational results with optical data from other labs on the same reaction, we have constructed an overall working model for the dioxygen reduction reaction and have calculated concentration/time profiles for key intermediates. As opposed to most O 2 metabolizing enzymes, these calculations indicate that the oxidase/O 2 reaction is under proton control, which allows transient intermediates to build to detectable concentrations. We have linked this behavior to the proton-pump function of the enzyme and have postulated that proton control allows tight coupling between the oxygen chemistry and the proton translocations it drives. Recent findings from several laboratories have shown that a direct connection can be made between the intermediates that occur during oxygen reduction by fully reduced and mixed-valence oxidase and in its reaction with peroxide. This requires a branching reaction at the peroxide level in the reaction sequence. This modification to the mechanism is presented. In this article, these findings are reviewed and the mechanism by which O 2 reduction is catalyzed by cytochrome oxidase is compared and contrasted with other O 2 -metabolizing heme enzymes. The continuing importance of vibrational spectroscopic approaches that rely on stable isotope substitution for mode identification is highlighted by reviewing recent developments in other laboratories on O 2 activation in the non-heme iron class of oxygen-metabolizing enzymes. This group of catalysts includes ribonucleotide reductase, methane monooxygenase, and fatty acid Δ 9 -desaturase and has been recognized as a distinct, oxygen-metabolizing enzyme class only recently. Finally, recent inorganic model compound work on O 2 activation is briefly summarized.

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.