Cytochrome P450 proteins (CYP450s) are membrane-associated haem proteins that metabolize physiologically important compounds in many species of microorganisms, plants and animals. Mammalian CYP450s recognize and metabolize diverse xenobiotics such as drug molecules, environmental compounds and pollutants. Human CYP450 proteins CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A4 are the major drug-metabolizing isoforms, and contribute to the oxidative metabolism of more than 90% of the drugs in current clinical use. Polymorphic variants have also been reported for some CYP450 isoforms, which has implications for the efficacy of drugs in individuals, and for the co-administration of drugs. The molecular basis of drug recognition by human CYP450s, however, has remained elusive. Here we describe the crystal structure of a human CYP450, CYP2C9, both unliganded and in complex with the anti-coagulant drug warfarin. The structure defines unanticipated interactions between CYP2C9 and warfarin, and reveals a new binding pocket. The binding mode of warfarin suggests that CYP2C9 may undergo an allosteric mechanism during its function. The newly discovered binding pocket also suggests that CYP2C9 may simultaneously accommodate multiple ligands during its biological function, and provides a possible molecular basis for understanding complex drug-drug interactions.

Cytochromes P450 (P450s) metabolize a wide range of endogenous compounds and xenobiotics, such as pollutants, environmental compounds, and drug molecules. The microsomal, membrane-associated, P450 isoforms CYP3A4, CYP2D6, CYP2C9, CYP2C19, CYP2E1, and CYP1A2 are responsible for the oxidative metabolism of more than 90% of marketed drugs. Cytochrome P450 3A4 (CYP3A4) metabolizes more drug molecules than all other isoforms combined. Here we report three crystal structures of CYP3A4: unliganded, bound to the inhibitor metyrapone, and bound to the substrate progesterone. The structures revealed a surprisingly small active site, with little conformational change associated with the binding of either compound. An unexpected peripheral binding site is identified, located above a phenylalanine cluster, which may be involved in the initial recognition of substrates or allosteric effectors.

The crystal structure and spectroscopic properties of the periplasmic penta-heme cytochrome c nitrite reductase (NrfA) of Escherichia coli are presented. The structure is the first for a member of the NrfA subgroup that utilize a soluble penta-heme cytochrome, NrfB, as a redox partner. Comparison to the structures of Wolinella succinogenes NrfA and Sulfospirillum deleyianum NrfA, which accept electrons from a membrane-anchored tetra-heme cytochrome (NrfH), reveals notable differences in the protein surface around heme 2, which may be the docking site for the redox partner. The structure shows that four of the NrfA hemes (hemes 2-5) have bis-histidine axial heme-Fe ligation. The catalytic heme-Fe (heme 1) has a lysine distal ligand and an oxygen atom proximal ligand. Analysis of NrfA in solution by magnetic circular dichroism (MCD) suggested that the oxygen ligand arose from water. Electron paramagnetic resonance (EPR) spectra were collected from electrochemically poised NrfA samples. Broad perpendicular mode signals at g similar 10.8 and 3.5, characteristic of weakly spin-coupled S = 5/2, S = 1/2 paramagnets, titrated with E(m) = -107 mV. A possible origin for these are the active site Lys-OH(2) coordinated heme (heme 1) and a nearby bis-His coordinated heme (heme 3). A rhombic heme Fe(III) EPR signal at g(z) = 2.91, g(y) = 2.3, g(x) = 1.5 titrated with E(m) = -37 mV and is likely to arise from bis-His coordinated heme (heme 2) in which the interplanar angle of the imidazole rings is 21.2. The final two bis-His coordinated hemes (hemes 4 and 5) have imidazole interplanar angles of 64.4 and 71.8. Either, or both, of these hemes could give rise to a "Large g max" EPR signal at g(z)() = 3.17 that titrated at potentials between -250 and -400 mV. Previous spectroscopic studies on NrfA from a number of bacterial species are considered in the light of the structure-based spectro-potentiometric analysis presented for the E. coli NrfA.

The molybdenum−iron (MoFe) protein of nitrogenase contains two unique metalloclusters called P-cluster [8Fe-7S] and M-center (FeMo cofactor, [7Fe-9S-Mo-homocitrate]). Using samples containing M-centers selectively enriched with 57Fe (57M56P), we have studied three M-center states with Mössbauer spectroscopy. The results are as follows. A detailed analysis of the Mössbauer spectra of the S = 3/2 state MN recorded in applied fields up to 8.0 T has revealed the features of the seventh Fe site which had eluded previous Mössbauer and ENDOR studies. This site has unusually small and anisotropic magnetic hyperfine interactions (A iso ≈ −4 MHz). Our studies have also revealed that the spectroscopic component previously labeled B 1 represents two equivalent Fe sites. Six of the M-center irons are trigonally coordinated to bridging sulfides; their unusual isomer shifts are discussed with particular reference to a trigonally coordinated Fe(II) thiolate complex synthesized by Power and co-workers (Inorg. Chem. 1995, 34, 1815−1822). The unusually low isomer shifts (δav = 0.41 mm/s) of MN suggest that the core of the M-center is (formally) best described as (Mo4+-3Fe3+-4Fe2+). The turnover complex MR is one electron further reduced than MN. While δav changes by 0.06 mm/s between the one-electron oxidized state MOX and MN, only a small change in δav, 0.02 mm/s, is observed between MN and MR. Moreover, spectra of the integer-spin state MR taken in strong applied magnetic fields are quite similar to those observed for MN, suggesting that the 7-Fe segment of the M-center has the same spin structure in both states. These observations suggest that the reduction MN → MR is associated mainly with the molybdenum site. In a preliminary experiment, we have also observed reduction of the M-cluster (ca. 40%) by irradiating a 57M56P sample at 77 K in a synchrotron X-ray beam. The radiolytically reduced state, MI, has integer electronic spin S ≥ 1, and its reduction appears to be centered on the iron components of the cluster.

Inhibitors of the molecular chaperone heat shock protein 90 (Hsp90) are currently generating significant interest in clinical development as potential treatments for cancer. In a preceding publication (DOI: 10.1021/jm100059d ) we describe Astex's approach to screening fragments against Hsp90 and the subsequent optimization of two hits into leads with inhibitory activities in the low nanomolar range. This paper describes the structure guided optimization of the 2,4-dihydroxybenzamide lead molecule 1 and details some of the drug discovery strategies employed in the identification of AT13387 (35), which has progressed through preclinical development and is currently being tested in man.

The cytochrome c nitrite reductases perform a key step in the biological nitrogen cycle by catalyzing the six-electron reduction of nitrite to ammonium. Graphite electrodes painted with Escherichia coli cytochrome c nitrite reductase and placed in solutions containing nitrite (pH 7) exhibit large catalytic reduction currents during cyclic voltammetry at potentials below 0 V. These catalytic currents were not observed in the absence of cytochrome c nitrite reductase and were shown to originate from an enzyme film engaged in direct electron exchange with the electrode. The catalytic current-potential profiles observed on progression from substrate-limited to enzyme-limited nitrite reduction revealed a fingerprint of catalytic behavior distinct from that observed during hydroxylamine reduction, the latter being an alternative substrate for the enzyme that is reduced to ammonium in a two electron process. Cytochrome c nitrite reductase clearly interacts differently with these two substrates. However, similar features underlie the development of the voltammetric response with increasing nitrite or hydroxylamine concentration. These features are consistent with coordinated two-electron reduction of the active site and suggest that the mechanisms for reduction of both substrates are underpinned by common rate-defining processes.

The ability to fix dinitrogen is restricted to a small but diverse group of procaryotes that contain the nitrogenase system. This system is composed of two proteins called the Fe protein and the MoFe protein; the latter contains the site of N 2 binding and reduction. 1,2 The Fe protein contains a single Fe 4 S 4 cluster bridged between two identical subunits each of which has a single binding site for MgATP. It is the only known reductant capable of reducing the MoFe protein such that the latter can reduce substrates. Our current understanding of nitrogenase is based on in vitro experiments with purified nitrogenase proteins using dithionite as the electron donor. Under these conditions the Fe 4 S 4 cluster shuttles between the 2+ and 1+ oxidation states.In 1994, Watt and Reddy 3 reported that the [Fe 4 S 4 ] 1+ Fe protein could be reversibly reduced (E°′ ) -460 mV vs SHE) to the all-ferrous [Fe 4 S 4 ] 0 state. This reaction was reported to be pH independent between pH 7.0 and 8.0. [Fe 4 S 4 ] 0 could be produced using methylviologen, Ti(III)citrate, or the physiological electron donor flavodoxin as reductants but could not be generated when dithionite was used as a reductant. 1,3,4 The proposal for the formation of an [Fe 4 S 4 ] 0 state was based on two main lines of evidence. 3 First, Coulometric reduction of the [Fe 4 S 4 ] 1+ state of the Fe protein showed that a second electron could be added to the protein but did not show whether the reduction was metal-centered. 5 Second, the two-electron reduced Fe protein did not exhibit the S ) 1 / 2 EPR signal that arises from the [Fe 4 S 4 ] 1+ state, but that signal could be elicited by adding 1 equiv of oxidant to the fully reduced protein. These observations suggest, but do not prove, the unprecedented formation 6 of a protein-bound all-ferrous Fe 4 S 4 cluster.For the Mössbauer and EPR studies we produced the twoelectron reduced form of the Fe protein by treating 57 Fe-enriched Azotobacter Vinelandii Fe protein (AV2) with Ti(III) citrate. 8 Figure 1 shows two Mössbauer spectra of Ti(III) citrate-reduced AV2. The zero-field spectra exhibit quadrupole doublets down to 4.2 K. Preliminary analysis of the whole data set (30 spectra) revealed four distinct sites, all with isomeric shift δ ) 0.68 mm/s. The simulation of Figure 1A assumes four doublets of equal intensity with ∆E Q ) 1.25, 1.40, 1.75, and 3.08 mm/s. The absence of magnetic features in the 4.2 K zero-field spectrum strongly suggests that the Fe 4 S 4 cluster has integer or zero spin. The isomer shift, δ, is an excellent indicator for the oxidation state of an Fe 4 S 4 cluster; typically, the average value of δ increases by 0.10-0.12 mm/s per electron added. Table 1 shows that the isomeric shifts of all iron sites are larger than those of the ferrous pair (δ ) 0.59 mm/s 12 ) of the S ) 1 / 2 state of [Fe 4 S 4 ] 1+ AV2. From these observations we conclude that all iron sites of the cluster are ferrous.As shown in Figure 1B, a weak applied magnetic field elicits substantial 57 Fe magnetic hyperfine inter...

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