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Candida albicans phosphomannose isomerase (PMI) is a zinc metalloprotein of known crystal structure. When heterologously overexpressed in Escherichia coli, a blue protein that contains tip to 0.5 iron atoms/PMI molecule could be isolated, with absorption maxima at 420 nm and 680 nm. These bands are reminiscent of ferric catecholate complexes, an assignment that has been confirmed by resonance Raman spectroscopy, and by reaction with Arnow's reagent, which is specific for the presence of 3,4-dihydroxy-phenylalanine (Dopa). After enzymatic digestion of blue PMI, a peptide with the sequence DPHAXISG was isolated corresponding to residues Asp283-Gly290 in the amino acid sequence of C. albicans PMI, where the unidentified residue X287 is encoded by a tyrosine codon. It is proposed that iron and oxygen bring about hydroxylation of Tyr287 in PMI and that Fe(III) subsequently chelates the Dopa residue to give the characteristic absorption spectrum. The EPR spectrum of the blue protein suggests three iron environments in the protein, two in axial environments with EID values approximately equal to 0.06 and 0.12 and one rhombic species. The nature of the iron co-ordination sites is discussed with the help of model systems and by comparison with other blue non-heme iron proteins. [ABSTRACT FROM AUTHOR]

Identifies the iron-sulphur clusters in a ferredoxin from the archaeon Sulfolobus acidocaldarius. Monomer molecular mass; Electrospray ionization mass spectrometry; Magnetic circular dichroic spectroscopy; Thin film electrochemical method.

A spectroscopic study of an aconitase, AcnA, from Escherichia coli is presented. The amino acid sequence of AcnA has 53% identity with mammalian cytosolic aconitase (c-aconitase) which is the translational regulator known as iron regulatory factor (IRF). In the [3Fe-4S]+-containing, inactive state, AcaA displays an EPR signal which is not unlike the corresponding signal from mammalian mitochondrial aconitase (m-aconitase) but is even more similar to the signal form c-aconitase. This is perhaps related to the greater similarity of AcnA amino acid sequence with c-aconitase. Magnetic circular dichroism (MCD) spectroscopy has revealed that the electronic structure of the [3Fe-4S] cluster of AcnA must be similar to, but ot identical to that of m-aconitase. Whilst the[3Fe-4S] clusters form both of these enzymes display some features in their MCD spectra common to [3Fe-4S] clusters in general, their spectra overall are unique and indicate that the Fea atom of the [4Fe-4S] form is not the only unusual feature of the [Fe-S] clusters of aconites. Active [4Fe-4S]-containing AcnA can be reduced to yield an EPR signal due to a [4Fe-4S]+ cluster which is indistinguishable from the signals from the [4Fe-4S]+ cluster in the mammalian enzymes. However, in contrast to the mammalian enzymes, the EPR signals of the cluster in AcnA are not significantly perturbed upon the addition of substrate. Furthermore,the catalytic activity of [4Fe-4S]2+-containing AcnA is fivefold higher than that of m-aconitase. The mechanistic implications of these data are discussed. A novel S =1/2 EPR signal with g ≈2 was observed in AcnA upon treatment with EDTA. The species giving rise to this signal is proposed to be an intermediate in cluster deconstruction. [ABSTRACT FROM AUTHOR]

Reports on the spectroscopic and mutagenesis studies on the copper centre from the cytochrome-c oxidase complex of Paracoccus denitrificans. Characteristics of the cooper centre; Magnetic circular dichroism spectrum; Model involving terminal cysteine ligation.

A Mo(V) electron paramagnetic resonance (EPR) study of the periplasmic respiratory nitrate reductase of the denitrifying bacterium Thiosphaera pantotropha has revealed that the molybdenum centre of this enzyme is very similar to that in the assimilatory nitrate reductase of Azotobacter vinelandii but is somewhat different from that of the membrane-bound bacterial respiratory nitrate reductases such as those of Escherichia coli and Paracoccus denitrificans. We have identified the Mo(V) species most likely to be the catalytically relevant one and characterized two other sets of Mo(V) EPR signals. As well as exhibiting EPR signals with g values typical of bacterial molybdenum-containing reductases, molybdenum-hydroxylase-like EPR signals can be elicited in the nitrate reductase of T. pantotropha upon treatment with excess dithionite. The only other enzyme known to display this phenomenon is the periplasmic dimethylsulphoxide reductase of Rhodobacter capsulatus. A mechanism for the generation of these signals is proposed which invokes reduction of the pterin ring of the molybdenum cofactor linked to GMP from the dihydro to the tetrahydro state. The possibilities and implications of there being cysteine ligands to the molybdenum centres of these two enzymes are discussed. [ABSTRACT FROM AUTHOR]

Room-temperature (295 K) magnetic-circular­dichroism spectra at 280–2500 nm have been recorded for Escherichia coli cytochrome bo in its fast form (which has a g = 3.7 EPR signal and reacts rapidly with cyanide) and for its formate, fluoride, cyanide and hydrogen-peroxide derivatives. The spectra of all forms are dominated by signals from low-spin ferric heme b. These include a porphyrin-to-ferric ion charge-transfer transition in the near-infrared region (the near-infrared charge-transfer band) at 1610 nm. High-spin ferric heme o gives rise to a negative magnetic-circular-dichroism feature at 635, 642 and 625 nm (corresponding to a shoulder observed in the electronic absorption spectra) and a derivative charge-transfer feature at 1100, 1180 and 940 nm for the fast, formate and fluoride forms, respectively. The energies of these bands confirm that fluoride and formate are ligands to heme o. The energies of the analogous bands in the spectrum of fast cytochrome bo are typical tot high-spin ferric hemes with histidine and water axial ligands. Addition of cyanide ion to fast cytochrome bo causes a red shift in the position of the Soret absorption peak, from 406.5 nm to 413 nm, and results in the loss of the 635-nm feature form the magnetic-circular-dichroism spectrum and of the corresponding shoulder in the electronic absorption spectrum. In the magnetic-circular-dichroism spectrum, the intensities of the Soret and α.β bands are significantly increased. New near-infrared charge-transfer intensity is observed at 1000–2300 nm with a peak 2050 nm. These changes are interpreted as resulting from a high-spin to low-spin transition at ferric heme o brought about by the binding of cyanide ion. The energy of the near-infrared charge-transfer band suggests that the cyanide ion is bridged to the CuB of the binuclear site. Treatment of fast cytochrome bo with hydrogen peroxide also causes a red shift in the position of the Soret absorbance, to 412 nm. and a loss of the 625-nm absorption shoulder: Changes in the magnetic-circular-dichroism spectrum at 450–600 nm are observed, but there is no significant increase in the intensity of the magnetic-circular-dichroism Soret band and no new near-infrared charge-transfer bands are detected, ruling out a similar high-spin to low-spin transition at heme o. Subtraction of the contribution of low-spin ferric heme b from the magnetic-circular-dichroism spectrum reveals a spectrum characteristic of ferryl [Fe(IV)] heme o. [ABSTRACT FROM AUTHOR]

The enzymic reduction of nitrite takes place in a wide range of bacteria and is found to occur in denitrifying, assimilatory and dissimilatory pathways. In this review we describe the major molecular characteristics of the various enzymes employed in each of these processes. [ABSTRACT FROM AUTHOR]