Previously, it was shown that the CYP1(HAP1) gene product mediates the transcription of several oxygen‐regulated genes through a metabolic co‐effector, heme, in the yeast Saccharomyces cerevisiae. This study investigates the overproduction of the CYP1 protein when the CYP1(HAP1) gene is placed under the control of the GAL10‐CYC1 hybrid promoter (either at the locus of the CYP1(HAP1) gene or cloned in a high‐copy‐number plasmid). In these conditions, the CYP1 protein is detected by Western blot analysis and has a molecular mass in agreement with the open reading frame sequence. Band‐shift experiments show that the CYP1(HAP1) protein is able to interact specifically with its target sequences in vitro without addition of hemin, and forms a large complex with one or several unidentified factors denoted as X. Addition of hemin allows the formation of a new complex which has a lower molecular mass. The internal deletion of the seven repeated amino acid sequences containing the KCPVDH motif in the CYP1(HAP1) protein modifies the heme responsiveness phenomenon observed in vitro in the band‐shift experiments and in vivo in the transcription of the CYB2, CYC1, CYP3(CYC7) and ERG11 genes. On the basis of these data, we propose a new model for heme‐induced activation of the CYP1 protein.

Resonance Raman spectra of Hansenula anomala L-lactate:cytochrome c oxidoreductase (or flavocytochrome b2), of its cytochrome b2 core, and of a bis(imidazole) iron-protoporphyrin complex were obtained at the Soret preresonance from the oxidized and reduced forms. Raman contributions from both the isoalloxazine ring of flavin mononucleotide (FMN) and the heme b2 were observed in the spectra of oxidized flavocytochrome b2. Raman diagrams showing frequency differences of selected FMN modes between aqueous and proteic environments were drawn for various flavoproteins. These diagrams were closely similar for flavocytochrome b2 and for flavodoxins. This showed that the FMN structure must be very similar in both types of proteins, despite their very different proteic pockets. However, the electron density at this macrocycle was found to be higher in flavocytochrome b2 than in these electron transferases. No significant difference was observed between the heme structures in flavocytochrome b2 and in cytochrome b2 core. The porphyrin center-N(pyrrole) distances in the oxidized and reduced heme b2 were estimated to be 1.990 and 2.022 A from frequencies of porphyrin skeletal modes, respectively. The frequency of the vinyl stretching mode of protoporphyrin was found to be very affected in resonance Raman spectra of flavocytochrome b2 and of cytochrome b2 core (1634-1636 cm-1) relative to those observed in the spectra of iron-protoporphyrin [bis(imidazole)] complexes (1620 cm-1). These specificities were interpreted as reflecting a near coplanarity of the vinyl groups of heme b2 with the pyrrole rings to which they are attached. The low-frequency regions of resonance Raman indicated that the iron atoms of the four hemes b2 are in the porphyrin plane whatever their oxidation state. The histidine-Fe-histidine symmetric stretching mode was located at 205 cm-1 in the spectra of flavocytochrome b2 and of cytochrome b2 core. It was insensitive to the iron oxidation state and indicated strong Fe-His bonds in both states.

The gene coding for nitrite reductase of Pseudomonas aeruginosa has been cloned and its sequence determined. The coding region is 1707 bp long and contains information for a polypeptide chain of 568 amino acids. The sequence of the mature protein has been confirmed independently by extensive amino acid sequencing. The amino-terminus of the mature protein is located at Lys-26; the preceding 25 residue long extension shows the features typical of signal peptides. Therefore the enzyme is probably secreted into the periplasmic space. The mature protein is made of 543 amino acid residues and has a molecular mass of 60204 Da. The c-heme-binding domain, which contains the only two Cys of the molecule, is located at the amino-terminal region. Analysis of the protein sequence in terms of hydrophobicity profile gives results consistent with the fact that the enzyme is fully water soluble and not membrane bound; the most hydrophilic region appears to correspond to the c-heme domain. Secondary structure predictions are in general agreement with previous analysis of circular dichroic data.Nitrite reductase; Primary structure; Pre-protein; Cytochrome oxidase; (Pseudomonas aeruginosa)

The oxidized and semiquinone anion radical forms of flavin mononucleotide carried by flavocytochrome b2 and L-lactate monooxygenase have been studied by resonance Raman (RR) spectroscopy. The RR spectra of their oxidized forms are compared with previously published RR data on various flavins and flavoproteins. Taking as a support available X-ray crystallographic data on flavoproteins, we have found correlations between the frequencies of RR bands II (1575-1588 cm-1), III (1534-1557 cm-1), and X (1244-1266 cm-1) and the H-bonding environment and/or the structure of the flavin ring. The present RR data provide strong evidence that the electron density, the conformation, and the H-bonding environment of the oxidized flavin mononucleotide of flavocytochrome b2 and L-lactate monooxygenase are different. As far as the anionic semiquinone form of flavoproteins is concerned, the behavior of two bands observed at 1280-1300 and 1320-1350 cm-1 suggests that they have vibrational origins similar to those of RR bands II and III of oxidized compounds. On this basis, the differences in conformation and H-bonding environment of the isoalloxazine ring, observed for the oxidized form of flavocytochrome b2 and L-lactate monooxygenase, appear to be preserved upon one-electron reduction of the flavin. For both flavoproteins, the RR spectra of the semiquinone form are affected by pyruvate binding. The data are interpreted in the frame of a change in H-bonding interaction of the C4&dbd;O carbonyl group of the flavin without significant alteration of the isoalloxazine conformation. This modification in electrostatic interaction quantitatively accounts for the pyruvate-induced changes of the oxidized/semiquinone and semiquinone/reduced redox potentials of the flavoproteins. Considering the high homology in the flavin catalytic sites of flavocytochrome b2 and L-lactate monooxygenase, the observed differences in H-bonding environment and conformation of the FMN ring are related to the different biological functions of the two flavoproteins.