Cytochrome P450cam catalyzes the hydroxylation of camphor in a complex process involving two electron transfers (ETs) from the iron-sulfur protein putidaredoxin. The enzymatic control of the successive steps of catalysis is critical for a highly efficient reaction. The injection of the successive electrons is part of the control system. To understand the molecular interactions between putidaredoxin and cytochrome P450cam, we determined the structure of the complex both in solution and in the crystal state. Paramagnetic NMR spectroscopy using lanthanide tags yielded 446 structural restraints that were used to determine the solution structure. An ensemble of 10 structures with an RMSD of 1.3Å was obtained. The crystal structure of the complex was solved, showing a position of putidaredoxin that is identical with the one in the solution structure. The NMR data further demonstrate the presence of a minor state or set of states of the complex in solution, which is attributed to the presence of an encounter complex. The structure of the major state shows a small binding interface and a metal-to-metal distance of 16Å, with two pathways that provide strong electronic coupling of the redox centers. The interpretation of these results is discussed in the context of ET. The structure indicates that the ET rate can be much faster than the reported value, suggesting that the process may be gated.

Nitrile hydratase from Rhodococcus sp. N-771 is an ab heterodimer with a nonheme ferric iron in the catalytic center. In the catalytic center, aCys112 and aCys114 are modified to a cysteine sulfinic acid~Cys-SO 2 H! and a cysteine sulfenic acid~Cys-SOH!, respectively. To understand the function and the biogenic mechanism of these modified residues, we reconstituted the nitrile hydratase from recombinant unmodified subunits. The ab complex reconstituted under argon exhibited no activity. However, it gradually gained the enzymatic activity through aerobic incubation. ESI-LC0MS analysis showed that the anaerobically reconstituted ab complex did not have the modification of aCys112-SO 2 H and aerobic incubation induced the modification. The activity of the reconstituted ab complex correlated with the amount of aCys112-SO 2 H. Furthermore, ESI-LC0MS analyses of the tryptic digest of the reconstituted complex, removed of ferric iron at low pH and carboxamidomethylated without reduction, suggested that aCys114 is modified to Cys-SOH together with the sulfinic acid modification of aCys112. These results suggest that aCys112 and aCys114 are spontaneously oxidized to Cys-SO 2 H and Cys-SOH, respectively, and aCys112-SO 2 H is responsible for the catalytic activity solely or in combination with aCys114-SOH.

The nitrile hydratase (NHase) from Rhodococcus sp. N-771 is a photoreactive enzyme that is inactivated on nitrosylation of the non-heme iron center and activated on photo-dissociation of nitric oxide (NO). The nitrile hydratase operon consists of six genes encoding NHase regulator 2, NHase regulator 1, amidase, NHase alpha subunit, NHase beta subunit and NHase activator. We overproduced the NHase in Escherichia coli using a T7 expression system. The NHase was functionally expressed in E. coli only when the NHase activator encoded downstream of the beta subunit gene was co-expressed and the transformant was grown at 30 degrees C or less. A ligand cysteine, alphaCys112, of the recombinant NHase was also post-translationally modified to a cysteine-sulfinic acid similar to for the native NHase. Although another modification of alphaCys114 could not be identified because of the instability under acidic conditions, the recombinant NHase could be reversibly inactivated by nitric oxide.

Recent earth science studies have pointed out that massive acceleration of the global nitrogen cycle by anthropogenic addition of bio-available nitrogen has led to a host of environmental problems. Nitrous oxide (N(2)O) is a greenhouse gas that is an intermediate during the biological process known as denitrification. Copper-containing nitrite reductase (CuNIR) is a key enzyme in the process; it produces a precursor for N(2)O by catalysing the one-electron reduction of nitrite (NO2-) to nitric oxide (NO). The reduction step is performed by an efficient electron-transfer reaction with a redox-partner protein. However, details of the mechanism during the electron-transfer reaction are still unknown. Here we show the high-resolution crystal structure of the electron-transfer complex for CuNIR with its cognate cytochrome c as the electron donor. The hydrophobic electron-transfer path is formed at the docking interface by desolvation owing to close contact between the two proteins. Structural analysis of the interface highlights an essential role for the loop region with a hydrophobic patch for protein-protein recognition; it also shows how interface construction allows the variation in atomic components to achieve diverse biological electron transfers.

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Nitrile hydratase (NHase) from Rhodococcus N-771, which catalyzes hydration of nitriles to the corresponding amides, exhibits novel photosensitivity; in the dark, it is in the inactive form that binds an endogenous nitric oxide (NO) molecule at the non-heme iron center, and photodissociation of the NO activates the enzyme. NHase is also known to have a unique active site structure. Two cysteine ligands to the iron center, alphaCys112 and alphaCys114, are post-translationally modified to sulfinic acid (Cys-SO(2)H) and sulfenic acid (Cys-SOH), respectively, which are thought to play a crucial role in the catalytic reaction. Here, we have determined the protonation structures of these Cys-SO(2)H and Cys-SOH groups using Fourier transform infrared (FTIR) spectroscopy in combination with density functional theory (DFT) calculations. The light-induced FTIR difference spectrum of NHase between the dark inactive and light active forms exhibited two prominent signals at (1154-1148)/1126 and (1040-1034)/1019 cm(-1), which downshifted to 1141/1114 and 1026/1012 cm(-1), respectively, in the uniformly (34)S-labeled NHase. In addition, a minor signal at 915/908 cm(-1) also showed a considerable downshift upon (34)S labeling. These (34)S-sensitive signals were basically conserved in D(2)O buffer with only slight shifts. Vibrational frequencies of methanesulfenic acid (CH(3)SOH) and methanesulfinic acid (CH(3)SO(2)H), simple model compounds of Cys-SOH and Cys-SO(2)H, respectively, were calculated using the DFT method in both the protonated and deprotonated forms and in metal complexes. Comparison of the calculated frequencies and isotope shifts with the observed ones provided the assignment of the two major signals around 1140 and 1030 cm(-1) to the asymmetric and symmetric SO(2) stretching vibrations, respectively, of the S-bonded Cys-SO(2)(-) complex, and the assignment of the minor signal around 910 cm(-1) most likely to the SO stretch of the S-bonded Cys-SO(-) complex. These assignments and the small frequency shifts upon deuteration are consistent with the view that the deprotonated alphaCys112-SO(2)(-) and alphaCys114-SO(-) are hydrogen-bonded with the protons from betaArg56 and/or betaArg141, forming a reactive cavity at the interface of the alpha and beta subunits. There is further speculation that either of these groups is hydrogen bonded to a reactant water molecule, increasing its basicity to facilitate the nucleophilic attack on the nitrile substrate bound to the iron center.

Dissimilatory nitrite reductase (NIR) is a key enzyme in denitrification, catalyzing the first step that leads to gaseous products (NO, N 2O, and N2). We have determined the crystal structure of a Cu-containing NIR from a methylotrophic denitrifying bacterium, Hyphomicrobium denitrificans, at 2.2-Å resolution. The overall structure of this H. denitrificans NIR reveals a trigonal prism-shaped molecule in which a monomer consisting of 447 residues and three Cu atoms is organized into a unique hexamer (i.e., a tightly associated dimer of trimers). Each monomer is composed of an N-terminal region containing a Greek key ␤-barrel folding domain, cupredoxin domain I, and a C-terminal region containing cupredoxin domains II and III. Both cupredoxin domains I and II bind one type 1 Cu and are combined with a long loop comprising 31 amino acid residues. The type 2 Cu is ligated at the interface between domain II of one monomer and domain III of an adjacent monomer. Between the two trimeric C-terminal regions are three interfaces formed by an interaction between the domains I, and the type 1 Cu in the domain is required for dimerization of the trimer. The type 1 Cu in domain II functions as an electron acceptor from an electron donor protein and then transfers an electron to the type 2 Cu, binding the substrate to reduce nitrite to NO. The discussion of the intermolecular electron transfer process from cytochrome c 550 to the H. denitrificans NIR is based on x-ray crystallographic and kinetic results.denitrification ͉ electron transfer ͉ redox partner ͉ intermolecular interaction ͉ cupredoxin

Biodirected epitaxial nanodeposition of polymers was achieved on a template with an oriented molecular surface. Acetobacter xylinum synthesized a ribbon of cellulose I microfibrils onto a fixed, nematic ordered substrate of glucan chains with unique surface characteristics. The substrate directed the orientation of the motion due to the inverse force of the secretion during biosynthesis, and the microfibrils were aligned along the orientation of the molecular template. Using real-time video analysis, the patterns and rates of deposition were elucidated. Field emission scanning electron microscopy revealed that a strong molecular interaction allowed for the deposition of nascent biosynthesized 3.5-nm cellulose microfibrils with inter-microfibrillar spacings of 7-8 nm on the surface of the template. The cellulose was deposited parallel to the molecular orientation of the template. Directed cellulose synthesis and ordered movement of cells were observed only by using a nematic ordered substrate made from cellulose, and not from ordered crystalline cellulose substrates or ordered cellulose-related synthetic polymers such as polyvinyl alcohol. This unique relationship between directed biosynthesis and the ordered fabrication from the nano to the micro scales could lead to new methodologies for the design of functional materials with desired nanostructures. Interfacial surface structure and interaction of materials at the nanoscale have attracted much attention in the field of nanotechnology (1). Microbiological systems have been investigated as a microscale process (2); however, recent studies showing the unique interaction of biological systems with entirely synthetic molecular assemblies have prompted consideration of a new generation of approaches for controlled nanoassembly (3). We report on an interaction of a biological system with an artificially oriented molecular substrate. The preparation of such an ordered substrate recently was achieved by Kondo and coworkers (4, 5) by dissolving native cellulose and reprecipitating it in a unique manner to form a distinctive structure termed nematic ordered cellulose (NOC) (Fig. 1). This is a unique type of glucan chain association. The structure is highly ordered but not crystalline and therefore may have exclusive properties as a material product over the native form of crystalline cellulose. The unique surface of NOC can be used as a template for the construction of nanocomposites. The methods for producing NOC also have been applied to other biopolymers, e.g., chitin, xylan, their derivatives, and blends of these polymers, and these will be reported in future papers.Native biopolymer assembly has been shown to be a complex process involving two separate, but integrated, steps of polymerization and crystallization (6). In particular, cellulose has been shown to be assembled by a macromolecular complex of enzymes located on the cell surface (7). Nature has designed an efficient system for regulating the molecular weight, crystallinity, size, and shape of the nanostruct...