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Fumarate and nitrate reduction regulatory (FNR) proteins are bacterial transcription factors that coordinate the switch between aerobic and anaerobic metabolism. In the absence of [O.sub.2], FNR binds a [[4Fe-4S].sup.2+] cluster (ligated by Cys-20, 23, 29, 122) promoting the formation of a transcriptionally active dimer. In the presence of [O.sub.2], FNR is converted into a monomeric, non-DNA-binding form containing a [[2Fe-2S].sup.2+] cluster. The reaction of the [[4Fe-4S].sup.2+] cluster with [O.sub.2] has been shown to proceed via a 2-step process, an [O.sub.2]-dependent 1-electron oxidation to yield a [[3Fe-4S].sup.+] intermediate with release of I [Fe.sup.2+] ion, followed by spontaneous rearrangement to the [[2Fe-2S].sup.2+] form with release of 1 [Fe.sup.3+] and 2 [S.sup.2-] ions. Here, we show that replacement of Ser-24 by Arg, His, Phe, Trp, or Tyr enhances aerobic activity of FNR in vivo. The FNR-S24F protein incorporates a [[4Fe-4S].sup.2+] cluster with spectroscopic properties similar to those of FNR. However, the substitution enhances the stability of the [[4Fe-4S].sup.2+] cluster in the presence of [O.sub.2]. Kinetic analysis shows that both steps 1 and 2 are slower for FNR-S24F than for FNR. A molecular model suggests that step 1 of the FNR-S24F iron-sulfur cluster reaction with [O.sub.2] is inhibited by shielding of the iron ligand Cys-23, suggesting that Cys-23 or the cluster iron bound to it is a primary site of [O.sub.2] interaction. These data lead to a simple model of the FNR switch with physiological implications for the ability of FNR proteins to operate over different ranges of in vivo [O.sub.2] concentrations. gene regulation | iron-sulfur | oxygen sensing | oxidation | transcriptional regulation

The studies have shown that in both native and reconstituted [4Fe-4S] FNR, the reaction environment and the presence of [Fe.sup.2} and [Fe.sup.3} chelators have influenced the cluster conversion reactions. The sensitivity of FNR cluster conversion kinetics to chelators has disclosed mechanism by which the cell can fine-tune its response to the onset of aerobic conditions.

A study presents the biophysical characterization of the sterol demethylase P450 (CYP51), which is obtained from Mycobacterium tuberculosis (MtB) and is chromosomally located adjacent to a ferrodoxin (Fdx). The analysis reveals that despite a thermodynamic barrier, Fdx forms a productive electron transfer complex and reduces it to a specific rate, as heme iron reduction is found to act as a rate-limiting step.

The interaction of nitric oxide with cytochrome P450 BM3 from Bacillus megaterium is analyzed by spectroscopic techniques and enzyme assays in order to identify nitrated tyrosine residue(s) in the enzyme. Nitric oxide ligates tightly to the ferric heme iron, inducing large changes in each of the main visible bands of the heme and inhibiting the fatty acid hydroxylase function of the protein.

A detailed structural, spectroscopic and thermodynamic characterization of cytochrome P450 BioI is conducted and reported. Results demonstrate unexpected flexibility of the active site with the capacity to bind tightly to certain steroids and to bulky azole drugs, as well as long-chain fatty acids. The result also reveals that BioI is purified from Escherichia coli with palmitic acid bound, which explains its isolation in a mixed spin state.

Electron paramagnetic resonance spectra( EPR) of purified heterotrimeric dimethyl sulfide dehydrogenase reveal resonances arising from multiple redox centers and a b-type heme. The first EPR spectroscopic characterization of the molybdenum center of the enzyme is presented.

A magneto-optical study is undertaken of the mixed-valence single-molecule magnet [MnIV (sub 4)MnIII (sub 8) O12 L16] in which the ligands, L, are acetate (Mn12Ac) or the long-chain carboxylic acid, C14 H-29COOH(Mn12C15) that confers better solubility in organic solvents. The optical polarization and the selection rules for field magnetic circular dichroism (MCD) select a subset of molecular orientations with respect to the direction of field.

Research has been conducted on the EcDos protein, e heme-based sensor protein detecting ligands with a heme-binding PAS domain. The displaceable residue's nature in the EcDos heme-PAS domain has been investigated via different techniques including mutagenesis and magnetic circular dichroism and the details are presented.

Results reveal that two of the heterodimeric c-type cytochrome SoxAX heme irons exhibit thiolate/nitrogen coordination. The third heme contains methionine/histidine protein ligands.

The action of bacterial nitric oxide reductase (NOR) on nitric oxide and nitrous oxide plays an integral role in the completion of the two-electron process undergone by these compounds. NOR is considered a diverged member of the heme-copper oxidase group since it exhibits a distinct electronic absorption spectrum. Experimental data derived from a Pavacocus denitrificans strain suggest that the oxygen reducing activity manifested by heme-copper oxidases is significantly attributed to the substitution of copper for non-heme iron in the dinuclear center.

A study was conducted on the fully oxidized nitric oxide reductase (NOR) from Pseudomonas stutzeri. The findings indicate a spin-spin interaction between the heme and another paramagnet as well as an unusual near-infrared MCD. Moreover, the results suggest that NOR is structurally related to heme-copper oxidases and that it has a heme/non-heme iron spin-coupled pair at the active site. Thus, the nature of the metal ion paired with the heme in the active site may be essential in determining the specificity of these enzymes.

The properties of the cytochromes cdsub1 from Pseudomonas stutzeri and Thiosphaera pantotropha have been compared, using MCD and EPR techniques. The results are consistent with previous indications that enzymes from different sources do not have identical heme iron ligands in the oxidized state, indicating there there are several classes of cytochrome cdsub1.

The binding of azide to the fast cytochrome bo of Escherichia coli was analyzed by electronic absorption spectroscopy. Room temperature changes produced insignificant alterations in the binding of azide to the subunit I or CuB of cytochrome bo which is composed of three metal centers, two heme proteins and one copper(II) ions. Furthermore, the immediate ligands of heme o exhibited small energy shifts during the binding of the azide to the CuB subunit of quinol oxidase.

Bis-methionine coordinated cytochrome b562 proteins with or without covalently attached heme were generated and characterized by multiple circular dichroism and electron paramagnetic resonance spectroscopies, and protein electrochemistry. In the oxidized state, the single variant (H102M) was high-spin and was coordinated by a methionine ligand. Likewise, the double variant (R98C/H102M) was bis-methionine coordinated in the ferrous state. In the oxidized state, the protein transformed from a slow-spin to a high-spin species but did not exhibit the methionine coordination state.

The low-spin complexes of three ferric tetramesitylporphyrin (FeIIITMP) are investigated by low-temperature magnetic circular dichroism (MCD) spectroscopy. Complexes 1 and 2 have ground state electronic configurations of (dxy)2(dxz,dyz)3 although they exhibit near-infrared porphyrin(pi)-ferric(d) charge transfer (NIR-CT) bands of completely varied MCD intensities. Complex 3 has vertical ligands and axial EPR spectrum of abnormal g-values and low MCD transitions in NIR, ultraviolet and visible wavelengths, which are attributed to a new (dxz,dyz)4(dxy)1 ground state.

The variant form Trp51Ala of yeast cytochrome c peroxidase has three active site states, and conversion from one form to another is dependent on ionic strength, specific anions and temperature. The electronic and MCD spectra exhibited by LS1, the acidic form, indicates a low-spin, bis-histidine coordination for the iron in the heme. The neutral HS form indicates a high-spin heme Fe(III), while the LS2 form indicates a low-spin Fe(III) derivative. A model of the active site of the enzyme is proposed, based on the nuclear magnetic resonance spectroscopy and electron absorption studies.

A study was conducted on the tetraheme flavocytochrome c from Shewanella putrefaciens NCIMB400 with emphasis on the N-terminal cytochrome domain using electron paramagnetic resonance and magnetic circular dichroism. Stopped-flow measurements were also employed to investigate the presence of paired hemes. Results showed that the four hemes observed were low spin and had a nitrogeneous sixth ligand. Stopped-flow data further revealed that fumarate-dependent heme oxidation was biphasic.

Temperature dependence of the magnetic circular dichroism (MCD) intensity is seen for the nitrite-treated form of cytochrome [cd.sub.1] nitrate reductase. The radical species is shown to be either a protein-bound radical generated by a nitrite ion reacting with a heme group resulting in a one-electron oxidation of an amino acid residue or a heme [d.sub.1] [Fe.sup.III]NO complex.

The spectroelectrochemical studies of Escherichia coli NrfA have provided unprecedented resolution of its thermodynamic properties. These methodologies have also facilitated studies of other oxidoreductases for which catalytic protein film voltammetry (PFV) is defined.

A bacterial expression system is described for human tryptophan 2,3-dioxygenase (rhTDO) along with spectroscopic, kinetic and redox analyses. The studies have shown that the rhRDO is catalytically active in the ferric form and significant differences are present across the heme dioxygenase family.

The glutamine at position 263 in Escherichia coli NrfA was mutated to understand the roles of the active site calcium ion and coordinating glutamine residue in nitrite reduction to ammonia by cytochrome c nitrite reductase. The findings provided insight into important function of unusual Q263-calcium ion pair to increase substrate affinity through its role in stabilization of a network of hydrogen bonded water molecules in the active site heme distal ligand.