A. Grant Mauk

Iron core mineralisation in prokaryotic ferritins

BACKGROUND To satisfy their requirement for iron while at the same time countering the toxicity of this highly reactive metal ion, prokaryotes have evolved proteins belonging to two distinct sub-families of the ferritin family: the bacterioferritins (BFRs) and the bacterial ferritins (Ftns). Recently, Ftn homologues have also been identified and characterised in archaeon species. All of these prokaryotic ferritins function by solubilising and storing large amounts of iron in the form of a safe but bio-available mineral. SCOPE OF REVIEW The mechanism(s) by which the iron mineral is formed by these proteins is the subject of much current interest. Here we review the available information on these proteins, with particular emphasis on significant advances resulting from recent structural, spectroscopic and kinetic studies. MAJOR CONCLUSIONS Current understanding indicates that at least two distinct mechanisms are in operation in prokaryotic ferritins. In one, the ferroxidase centre acts as a true catalytic centre in driving Fe(2+) oxidation in the cavity; in the other, the centre acts as a gated iron pore by oxidising Fe(2+) and transferring the resulting Fe(3+) into the central cavity. GENERAL SIGNIFICANCE The prokaryotic ferritins exhibit a wide variation in mechanisms of iron core mineralisation. The basis of these differences lies, at least in part, in structural differences at and around the catalytic centre. However, it appears that more subtle differences must also be important in controlling the iron chemistry of these remarkable proteins.

Control of metalloprotein redox potentials: what does site-directed mutagenesis of hemoproteins tell us?

Abstract An overview of factors affecting metalloprotein redox potentials is given, together with an assessment of the role of site-directed mutagenesis of hemoproteins in investigating this topic and a description of several theoretically challenging hemoproteins. The conclusion is reached that a quantitative and experimentally testable description of metalloprotein redox potentials is not yet available.

Synthesis of indoleamine 2,3-dioxygenase inhibitory analogues of the sponge alkaloid exiguamine A.

Synthetic analogues of the sponge natural product exiguamine A (3) have been prepared and evaluated for their ability to inhibit indoleamine 2,3-dioxygenase in vitro.

Experimental and theoretical analysis of the interaction between cytochromec and cytochromeb5

Experimental and theoretical investigation of the interaction of cytochromec and cytochromeb5 performed over nearly twenty years has produced considerable insight into the manner in which these proteins recognize and bind to each other. The results of these studies and the experimental and theoretical strategies that have been developed to achieve these results have significant implications for understanding the behavior of similar complexes formed by more complex and less-well characterized electron transfer proteins. The current review provides a comprehensive summary and critical evaluation of the literature on which the current status of our understanding of the interaction of cytochromec and cytochromeb5 is based. The general issues related to the study of electron transfer complexes of this type are discussed and some new directions for future investigation of such systems are considered.

A cytochrome c variant resistant to heme degradation by hydrogen peroxide

BACKGROUND Cytochrome c has peroxidase-like activity and can catalyze the oxidation of a variety of organic substrates, including aromatic, organosulfur and lipid compounds. Like peroxidases, cytochrome c is inactivated by hydrogen peroxide. During this inactivation the heme prosthetic group is destroyed. RESULTS Variants of the iso-1-cytochrome c were constructed by site-directed mutagenesis and were found to be more stable in the presence of hydrogen peroxide than the wild type. No heme destruction was detected in a triple variant (Tyr67-->Phe/Asn52-->Ile/Cys102-->Thr) with the catalytic hydrogen peroxide concentration of 1 mM, even following the loss of catalytic activity, whereas both double variants Tyr67-->Phe/Cys102-->Thr and Asn52-->Ile/Cys102-->Thr showed a greater rate of peroxide-induced heme destruction than observed with the wild-type protein. CONCLUSIONS Heme destruction and catalytic inactivation are two independent processes. An internal water molecule (Wat166) is shown to be important in the heme destruction process. The absence of a protein radical in the resistant variant suggests that the protein radical is necessary in the heme destruction process, but presumably is not involved in the reactions leading up to the protein inactivation.

Analysis of vibrational spectra of multicomponent systems. Application to pH-dependent resonance Raman spectra of ferricytochrome c

Abstract A method is presented which is appropriate to analyse complex vibrational spectra of molecular systems including several components. The method which is related to global analysis is directed to determine the spectra of the individual components together with their relative contributions in a given set of vibrational spectra. The advantages of this approach are investigated by systematically analysing a series of artificially generated spectra. The applicability of this method to the analysis of experimental spectra is demonstrated in a resonance Raman study of a pH-dependent conformational equilibrium of cytochrome c .

Plectosphaeroic acids A, B, and C, indoleamine 2,3-dioxygenase inhibitors produced in culture by a marine isolate of the fungus Plectosphaerella cucumerina.

Laboratory cultures of the fungus Plectosphaerella cucumerina obtained from marine sediments collected in Barkley Sound, British Columbia, yielded the novel alkaloids plectosphaeroic acids A (1) to C (3). The alkaloids 1-3 are inhibitors of indoleamine 2,3-dioxygenase (IDO).

Biomimetic synthesis of the IDO inhibitors exiguamine A and B

Biomimetic synthesis is an attempt to assemble natural products along biosynthetic lines without recourse to the full enzymatic machinery of nature. We exemplify this with a total synthesis of exiguamine A and the newly isolated natural product exiguamine B. The most noteworthy feature of this work is an oxidative endgame drawing from the complex chemistry of catecholamines, which allows for ready access to a new class of nanomolar indoleamine-2,3-dioxygenase inhibitors.

Expression and characterization of a recombinant multi-copper oxidase: laccase IV from Trametes versicolor

Abstract A cDNA encoding LccIV, a previously uncharacterized laccase isozyme of the white-rot basidiomycete Trametes versicolor , was expressed in the methylotrophic yeast Pichia pastoris . The LccIV isozyme is not expressed by T. versicolor under normal culture conditions and the enzyme was, therefore, investigated to determine whether it had any unusual properties. The native signal peptide of LccIV directed efficient secretion and correct proteolytic processing of LccIV to the mature form, whereas, substitution with the Saccharomyces cerevisiae α-mating factor signal peptide led to retention of an additional tetrapeptide at the amino-terminus of the secreted enzyme and ∼25% lower specific activity in fermentor medium. Active LccIV was purified to homogeneity by sequential steps of ion-exchange, size-exclusion and hydrophobic interaction chromatography. The enzyme contains ∼25% N -linked glycans (∼40% total carbohydrate) and has an apparent molecular mass of ∼85 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and ∼100 kDa by size-exclusion chromatography, indicating a monomeric structure. A pH of 5.5 was optimal for oxidation of 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid). Thus, the LccIV isozyme appears to be similar in these respects to the laccase isozymes constitutively expressed by T. versicolor .

Site-directed mutagenesis improves the biocatalytic activity of iso-1-cytochrome c in polycyclic hydrocarbon oxidation

Abstract iso-1-Cytochrome c from Saccharomyces cerevisiae is able to oxidize polycyclic aromatic hydrocarbons (PAH) in the presence of hydrogen peroxide. Anthracene and pyrene are oxidized by yeast cytochrome c to form anthraquinone and 1,8-pyrenedione, respectively. Iso-1-cytochrome c from S. cerevisiae was modified by site-directed mutagenesis of Phe82 and Cys102. The Phe82 substitution significantly altered the kinetic behavior of the protein; Cys102 modification affected neither the kinetic nor the stability constant. The Gly82;Thr102 variant was 10 times more active and showed a catalytic efficiency 10-fold greater than the wild-type iso-1-cytochrome c. However, Phe82 variants showed lower stability against inactivation by hydrogen peroxide than the wild-type protein. These site-directed mutations did not significantly alter the stability and activity of the hemoprotein in increasing concentrations of tetrahydrofuran.