Phenol hydroxylase (PH) belongs to a family of bacterial multicomponent monooxygenases (BMMs) with carboxylate-bridged diiron active sites. Included are toluene/o-xylene (ToMO) and soluble methane (sMMO) monooxygenase. PH hydroxylates aromatic compounds, but unlike sMMO, it cannot oxidize alkanes despite having a similar dinuclear iron active site. Important for activity is formation of a complex between the hydroxylase and a regulatory protein component. To address how structural features of BMM hydroxylases and their component complexes may facilitate the catalytic mechanism and choice of substrate, we determined X-ray structures of native and SeMet forms of the PH hydroxylase (PHH) in complex with its regulatory protein (PHM) to 2.3 Å resolution. PHM binds in a canyon on one side of the (αβγ) 2 PHH dimer, contacting α-subunit helices A, E, and F ∼12 Å above the diiron core. The structure of the dinuclear iron center in PHH resembles that of mixed-valent MMOH, suggesting an Fe(II)Fe(III) oxidation state. Helix E, which comprises part of the iron-coordinating four-helix bundle, has more π-helical character than analogous E helices in MMOH and ToMOH lacking a bound regulatory protein. Consequently, conserved active site Thr and Asn residues translocate to the protein surface, and an ∼6 Å pore opens through the four-helix bundle. Of likely functional significance is a specific hydrogen bond formed between this Asn residue and a conserved Ser side chain on PHM. The PHM protein covers a putative docking site on PHH for the PH reductase, which transfers electrons to the PHH diiron center prior to O 2 activation, † This research was supported by National Institute of General Medical Sciences Grant GM32134 (S.J.L.) and the Italian Ministry of SUPPORTING INFORMATION AVAILABLE BMM hydroxylase and regulatory protein structures and their electrostatic surfaces (Figures S1 and S2), packing of the β-subunit Nterminus and a γ-subunit from an adjacent molecule in the PHH canyon ( Figure S3), comparison of known regulatory protein structures ( Figure S4), sequence alignments of the different regulatory proteins ( Figure S5), models of the PHH diiron center in electron density maps ( Figure S6), comparison of the hydroxylase zinc-binding sequences ( Figure S7), and electron density maps around helix F ( Figure S8). This material is available free of charge via the Internet at http://pubs.acs.org. NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2007 March 21. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscriptsuggesting that the regulatory component may function to block undesired reduction of oxygenated intermediates during the catalytic cycle. A series of hydrophobic cavities through the PHH α-subunit, analogous to those in MMOH, may facilitate movement of the substrate to and/or product from the active site pocket. Comparisons between the ToMOH and PHH structures provide insights into their substrate regiospecificities.Bacterial multicomponent monooxygenases...

Heme is a vital molecule for all life forms with heme being capable of assisting in catalysis, binding ligands, and undergoing redox changes. Heme-related dysfunction can lead to cardiovascular diseases with the oxidation of the heme of soluble guanylyl cyclase (sGC) critically implicated in some of these cardiovascular diseases. sGC, the main nitric oxide (NO) receptor, stimulates second messenger cGMP production, whereas reactive oxygen species are known to scavenge NO and oxidize/inactivate the heme leading to sGC degradation. This vulnerability of NO-heme signaling to oxidative stress led to the discovery of an NO-independent activator of sGC, cinaciguat (BAY 58 -2667), which is a candidate drug in clinical trials to treat acute decompensated heart failure. Here, we present crystallographic and mutagenesis data that reveal the mode of action of BAY 58 -2667. The 2.3-Å resolution structure of BAY 58 -2667 bound to a heme NO and oxygen binding domain (H-NOX) from Nostoc homologous to that of sGC reveals that the trifurcated BAY 58 -2667 molecule has displaced the heme and acts as a heme mimetic. Carboxylate groups of BAY 58 -2667 make interactions similar to the heme-propionate groups, whereas its hydrophobic phenyl ring linker folds up within the heme cavity in a planar-like fashion. BAY 58 -2667 binding causes a rotation of the ␣F helix away from the heme pocket, as this helix is normally held in place via the inhibitory His 105 -heme covalent bond. The structure provides insights into how BAY 58 -2667 binds and activates sGC to rescue heme-NO dysfunction in cardiovascular diseases.

The three-dimensional X-ray crystal structure of the ‘rare cutting’ type II restriction endonuclease SgrAI bound to cognate DNA is presented. SgrAI forms a dimer bound to one duplex of DNA. Two Ca2+ bind in the enzyme active site, with one ion at the interface between the protein and DNA, and the second bound distal from the DNA. These sites are differentially occupied by Mn2+, with strong binding at the protein–DNA interface, but only partial occupancy of the distal site. The DNA remains uncleaved in the structures from crystals grown in the presence of either divalent cation. The structure of the dimer of SgrAI is similar to those of Cfr10I, Bse634I and NgoMIV, however no tetrameric structure of SgrAI is observed. DNA contacts to the central CCGG base pairs of the SgrAI canonical target sequence (CR|CCGGYG, | marks the site of cleavage) are found to be very similar to those in the NgoMIV/DNA structure (target sequence G|CCGGC). Specificity at the degenerate YR base pairs of the SgrAI sequence may occur via indirect readout using DNA distortion. Recognition of the outer GC base pairs occurs through a single contact to the G from an arginine side chain located in a region unique to SgrAI.