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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]

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]

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]

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]