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We have applied resonance Raman spectroscopy to investigate the properties of the dinuclear center of oxidized, reduced, and NO-bound nitric-oxide reductase from Paracoccus denitrificans. The spectra of the oxidized enzyme show two distinct nu(as)(Fe-O-Fe) modes at 815 and 833 cm(-1) of the heme/non-heme diiron center. The splitting of the Fe-O-Fe mode suggests that two different conformations (open and closed) are present in the catalytic site of the enzyme. We find evidence from deuterium exchange experiments that in the dominant conformation (833 cm(-1) mode, closed), the Fe-O-Fe unit is hydrogen-bonded to distal residue(s). The ferric nitrosyl complex of nitric-oxide reductase exhibits the nu(Fe(3+)-NO) and nu(N-O) at 594 and 1904 cm(-1), respectively. The nitrosyl species we detect is photolabile and can be photolyzed to generate a new form of oxidized enzyme in which the proximal histidine is ligated to heme b(3), in contrast to the resting form. Photodissociation of the NO ligand yields a five-coordinate high-spin heme b(3). Based on the findings reported here, the structure and properties of the dinuclear center of nitric- oxide reductase in the oxidized, reduced, and NO-bound form as well as its photoproduct can be described with certainty.

Nitrous-oxide reductases (N2OR) catalyze the two-electron reduction of N(2)O to N(2). The crystal structure of N2ORs from Pseudomonas nautica (Pn) and Paracoccus denitrificans (Pd) were solved at resolutions of 2.4 and 1.6 A, respectively. The Pn N2OR structure revealed that the catalytic CuZ center belongs to a new type of metal cluster in which four copper ions are liganded by seven histidine residues. A bridging oxygen moiety and two other hydroxide ligands were proposed to complete the ligation scheme (Brown, K., Tegoni, M., Prudencio, M., Pereira, A. S., Besson, S., Moura, J. J. G., Moura, I., and Cambillau, C. (2000) Nat. Struct. Biol. 7, 191-195). However, in the CuZ cluster, inorganic sulfur chemical determination and the high resolution structure of Pd N2OR identified a bridging inorganic sulfur instead of an oxygen. This result reconciles the novel CuZ cluster with the hitherto puzzling spectroscopic data.

In cytochrome c oxidase the C-terminal part of subunit II is outside the membrane and contains a copper center called CuA. We have expressed this domain of the Paracoccus denitrificans oxidase in a soluble form. Data obtained by quantitative copper-to-protein measurements, electrospray mass spectrometry, and electron paramagnetic resonance spectroscopy show that the center contains two copper atoms probably in a mixed valence configuration. Its absorbance spectrum is similar to that of the copper center A in nitrous oxide reductase. The EPR spectrum suggests that the center in the soluble protein is closely related to the native CuA site in the cytochrome oxidase complex. However, it seems likely that the copper center in the soluble domain is more exposed to the aqueous milieu than in the intact complex because its absorbance and EPR spectra are sensitive to pH. At alkaline pH one of the coppers in the site acquires type-2 character, indicating that it may be coordinated to a new ligand. The pK of this reversible change is about 8.2. The CuA-binding fragment is able to oxidize cytochrome c.

Cytochrome c oxidase contains a copper center, CuA, which is involved in electron transfer from cytochrome c to the oxygen-reducing active site. This center is distinct from types 1, 2, and 3 copper sites and related only to a purple copper center in nitrous oxide reductase. At present it is not clear whether this site is mononuclear or is comprised of two copper atoms in a mixed valence (Cu(I)-Cu(II)) configuration. Here we use a model of CuA, engineered into a structurally related but initially copperless protein, to study the structure of this copper center. The results from biochemical analysis, site-directed mutagenesis, and electrospray mass spectrometry support the binuclear model. Two cysteines, two histidines, and one methionine are the major ligands of two coppers. Substitution of these residues results in either a complete loss of color or dramatic changes in the absorbance spectrum. In contrast, substitution of the invariant glutamate residue, which is located between the copper-binding cysteines, leads to a minor perturbation of the optical spectrum.

We report the isolation and characterization of six overlapping cDNA clones that provide the first and complete amino acid sequence of the human laminin B1 chain. The cDNA clones cover 5613 nucleotides with 5358 nucleotides in an open reading frame encoding 1786 amino acids, including a 21-residue signal peptide-like sequence. Sequence analysis demonstrated the presence of two types of internal homology repeats that were found in clusters within the polypeptide chain. The type A repeats contain about 50 amino acids of which 8 are cysteine. These repeats are present in two clusters toward the NH2-terminal end of the chain and are separated from each other by about 220 amino acids. The two clusters contain five and eight consecutive repeats each. There are two copies of consecutive type B repeats of about 40 amino acids close to the COOH-terminal end. Computer analysis of the amino acid sequence of the B1 chain revealed the presence of structurally distinct domains that contain cysteine-rich repeats, globular regions, and helical structures. Using somatic cell hybrid methodology and in situ hybridization to metaphase chromosomes it was established that the human laminin B1 gene (LAMB1) is located in the q22 region of chromosome 7.