Influenza A virus is a major human and animal pathogen with the potential to cause catastrophic loss of life. The virus reproduces rapidly, mutates frequently and occasionally crosses species barriers. The recent emergence in Asia of avian influenza related to highly pathogenic forms of the human virus has highlighted the urgent need for new effective treatments. Here we demonstrate the importance to viral replication of a subunit interface in the viral RNA polymerase, thereby providing a new set of potential drug binding sites entirely independent of surface antigen type. No current medication targets this heterotrimeric polymerase complex. All three subunits, PB1, PB2 and PA, are required for both transcription and replication. PB1 carries the polymerase active site, PB2 includes the capped-RNA recognition domain, and PA is involved in assembly of the functional complex, but so far very little structural information has been reported for any of them. We describe the crystal structure of a large fragment of one subunit (PA) of influenza A RNA polymerase bound to a fragment of another subunit (PB1). The carboxy-terminal domain of PA forms a novel fold, and forms a deep, highly hydrophobic groove into which the amino-terminal residues of PB1 can fit by forming a 3(10) helix.

Influenza virus RNA-dependent RNA polymerase is a multi-functional heterotrimer, which uses a 'cap-snatching' mechanism to produce viral mRNA. Host cell mRNA is cleaved to yield a cap-bearing oligonucleotide, which can be extended using viral genomic RNA as a template. The cap-binding and endonuclease activities are only activated once viral genomic RNA is bound. This requires signalling from the RNA-binding PB1 subunit to the cap-binding PB2 subunit, and the interface between these two subunits is essential for the polymerase activity. We have defined this interaction surface by protein crystallography and tested the effects of mutating contact residues on the function of the holo-enzyme. This novel interface is surprisingly small, yet, it has a crucial function in regulating the 250 kDa polymerase complex and is completely conserved among avian and human influenza viruses.

Structures of nitric oxide reductase (NOR) in the ferric resting and the ferrous CO states have been solved at 2.0 A resolution. These structures provide significant new insights into how NO is reduced in biological systems. The haem distal pocket is open to solvent, implicating this region as a possible NADH binding site. In combination with mutagenesis results, a hydrogen-bonding network from the water molecule adjacent to the iron ligand to the protein surface of the distal pocket through the hydroxyl group of Ser 286 and the carboxyl group of Asp 393 can be assigned to a pathway for proton delivery during the NO reduction reaction.

Nitric oxide reductase from the denitrifying fungus Fusarium oxysporum catalyzes the reduction of NO to N 2 O [Nakahara, K., et al. J. Biol. Chem. 1993, 268, 8350-8355]. Since this enzyme belongs to the cytochrome P450 superfamily [Kizawa, H., et al. J. Biol. Chem. 1991, 266, 10632-10637], it is called cytochrome P450nor (P450nor), but does not exhibit monooxygenation activity. In the present study, we examine the coordination structure of the heme iron in P450nor in the ferric-NO form by using infrared, resonance Raman, and X-ray absorption (EXAFS ) extended X-ray absorption fine structure) spectroscopies, since the ferric-NO complex is a first intermediate in the NO reduction cycle by P450nor [Shiro, Y, et al J. Biol. Chem. 1995, 270, 1617-1623. We compared the data obtained with those for the d-camphor-bound form of Pseudomonas putida camphor hydroxylase cytochrome P450cam (P450cam), a typical model of the monooxygenase. From the vibrational spectroscopic measurements, we found that the Fe-bound N-O stretching frequency (ν(N-O)) occurred at 1851 cm -1 and the Fe-NO stretching frequency (ν(Fe-NO)) at 530 cm -1 for P450nor, while those at 1806 and 522 cm -1 were observed for P450cam, respectively. The assignments were confirmed by the 15 NO substituting effect on these vibrational frequencies. These results indicated that NO binds to the ferric iron in P450nor stronger than in P450cam. Support for this was provided from the EXAFS study, which gave an Fe-N NO bond distance of 1.66 ( 0.02 Å for P450nor and 1.76 ( 0.02 Å for P450cam. These spectroscopic results suggest that, compared with P450cam, the lower steric hindrance and/or the difference in the electrostatic interactions of the ligand NO with its surroundings facilitates the donation of the NO 2pπ* electron to the iron 3dπ orbital, resulting in the strengthening of the Fe-NO and the N-O bonds of P450nor. The vibrational spectral observation of the ferrous-CO complex of P450nor supported this suggestion. This configuration can reduce the electron density on the NO ligand in P450nor, which is seemingly relevant to the NO reduction reactivity of P450nor.

The benzylindazole compound YC-1 has been shown to activate soluble guanylate cyclase by increasing the sensitivity toward NO and CO. Here we report the action of YC-1 on the coordination of CO-and NO-hemes in the enzyme and correlate the events with the activation of enzyme catalysis. A single YC-1-binding site on the heterodimeric enzyme was identified by equilibrium dialysis. To explore the affect of YC-1 on the NO-heme coordination, the six-coordinate NO complex of the enzyme was stabilized by dibromodeuteroheme substitution. Using the dibromodeuteroheme enzyme, YC-1 converted the six-coordinate NO-heme to a five-coordinate NOheme with a characteristic EPR signal that differed from that in the absence of YC-1. These results revealed that YC-1 facilitated cleavage of the proximal His-iron bond and caused geometrical distortion of the five-coordinate NO-heme. Resonance Raman studies demonstrated the presence of two iron-CO stretch modes at 488 and 521 cm ؊1 specific to the YC-1-bound CO complex of the native enzyme. Together with the infrared C-O stretching measurements, we assigned the 488-cm ؊1 band to the iron-CO stretch of a six-coordinate CO-heme and the 521-cm ؊1 band to the iron-CO stretch of a fivecoordinate CO-heme. These results indicate that YC-1 stimulates enzyme activity by weakening or cleaving the proximal His-iron bond in the CO complex as well as the NO complex.

The pre-mRNA splicing reaction of eukaryotic cells has to be carried out extremely accurately, as failure to recognize the splice sites correctly causes serious disease. The small subunit of the U2AF heterodimer is essential for the determination of 3 ′ splice sites in pre-mRNA splicing, and several single-residue mutations of the U2AF small subunit cause severe disorders such as myelodysplastic syndromes. However, the mechanism of RNA recognition is poorly understood. Here we solved the crystal structure of the U2AF small subunit (U2AF23) from fission yeast, consisting of an RNA recognition motif (RRM) domain flanked by two conserved CCCH-type zinc fingers (ZFs). The two ZFs are positioned side by side on the β sheet of the RRM domain. Further mutational analysis revealed that the ZFs bind cooperatively to the target RNA sequence, but the RRM domain acts simply as a scaffold to organize the ZFs and does not itself contact the RNA directly. This completely novel and unexpected mode of RNA-binding mechanism by the U2AF small subunit sheds light on splicing errors caused by mutations of this highly conserved protein.

Fungal nitric-oxide reductase (NOR) is a heme enzyme that catalyzes the reduction of NO to Nitric oxide (NO)1 serves as a messenger molecule for a variety of biological functions, including neurotransmission, vascular relaxation, and the inhibition of platelet aggregation. In mammalian systems, NO is generated from L-arginine and molecular oxygen (O 2 ), via catalysis by heme-enzyme nitricoxide synthase, whose crystal structure has recently been reported (1-3). Subsequently, the generated NO binds to the heme iron of soluble guanylate cyclase activating the conversion of GTP to cGMP. In addition, the crystal structures of the heme-based NO transport protein, nitrophorin, of a blood sucking insect were recently reported (4,5). NO is also a potential ligand (inhibitor) of many hemoproteins such as myoglobin, hemoglobin, and peroxidase (6 -8). Despite the close relation of NO to hemoproteins, only a small amount of structural information is available for NO adduct of hemoproteins. It is particularly noteworthy that much less is known concerning the ferric-NO (Fe 3ϩ -NO) complex of hemoproteins, and only one crystal structure of cytochrome c peroxidase by Poulos and co-workers (8, 9) and two of the ferric-porphyrin model compounds, Fe Fungal nitric oxide reductase (NOR), a heme enzyme, is involved in the denitrification process by the fungus Fusarium oxysporum (11). In this process, NO is produced from the reduction of NO 2 Ϫ catalyzed by nitrite reductase, which represents an additional NO generating biological system. In order to detoxify the generated NO, fungal NOR converts NO to N 2 O by the reaction (11),Based on spectroscopic and kinetic studies of this reaction, we proposed that the overall enzymatic reaction (Scheme 1) consists of three chemical reactions, Schemes 2-4 (12);

The nature of the metal-proximal base bond of soluble guanylate cyclase from bovine lung was examined by EPR spectroscopy. When the ferrous enzyme was mixed with NO, a new species was transiently produced and rapidly converted to a five-coordinate ferrous NO complex. The new species exhibited the EPR signal of sixcoordinate ferrous NO complex with a feature of histidine-ligated heme. The histidine ligation was further examined by using the cobalt protoporphyrin IX-substituted enzyme. The Co 2؉ -substituted enzyme exhibited EPR signals of a broad g Ќ Ќ component and a g ሻ ሻ component with a poorly resolved triplet of 14 N superhyperfine splittings, which was indicative of the histidine ligation. These EPR features were analogous to those of ␣-subunits of Co 2؉ -hemoglobin in tense state, showing a tension on the iron-histidine bond of the enzyme. The binding of NO to the Co 2؉ -enzyme markedly stimulated the cGMP production by forming the five-coordinate NO complex. We found that N 3 ؊ elicited the activation of the ferric enzyme by yielding five-coordinate high spin N 3 ؊ heme. These results indicated that the activation of the enzymes was initiated by NO binding to the metals and proceeded via breaking of the metal-histidine bonds, and suggested that the iron-histidine bond in the ferric enzyme heme was broken by N 3 ؊ binding.