Myles R. Cheesman

Nitric oxide in bacteria: synthesis and consumption

5. Nitric oxide signalling in bacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 466 5.1. Denitrifying bacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 466 5.2. Pathogenic bacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 467 5.3. Parallels with eukaryotic systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 468

Spectroscopic characterization of a novel multihemec-type Cytochrome widely implicated in bacterial electron transport

NapC is a member of a family of bacterial membrane-anchored tetra-heme c-type cytochromes that participate in a number of respiratory electron transport pathways. They are postulated to mediate electron transfer between membrane quinols/quinones and soluble periplasmic enzymes. The water-soluble heme domain of NapC has been expressed as a periplasmic protein. Mediated redox potentiometry and characterization by UV-visible, magnetic circular dichroism, and electron paramagnetic resonance spectroscopies demonstrates that soluble NapC contains four low spin hemes, each with bis-histidine axial ligation and with midpoint reduction potentials of −56, −181, −207, and −235 mV.

Structural Basis for the Oxidation of Thiosulfate by a Sulfur Cycle Enzyme

Reduced inorganic sulfur compounds are utilized by many bacteria as electron donors to photosynthetic or respiratory electron transport chains. This metabolism is a key component of the biogeochemical sulfur cycle. The SoxAX protein is a heterodimeric c‐type cytochrome involved in thiosulfate oxidation. The crystal structures of SoxAX from the photosynthetic bacterium Rhodovulum sulfidophilum have been solved at 1.75 Å resolution in the oxidized state and at 1.5 Å resolution in the dithionite‐reduced state, providing the first structural insights into the enzymatic oxidation of thiosulfate. The SoxAX active site contains a haem with unprecedented cysteine persulfide (cysteine sulfane) coordination. This unusual post‐translational modification is also seen in sulfurtransferases such as rhodanese. Intriguingly, this enzyme shares further active site characteristics with SoxAX such as an adjacent conserved arginine residue and a strongly positive electrostatic potential. These similarities have allowed us to suggest a catalytic mechanism for enzymatic thiosulfate oxidation. The atomic coordinates and experimental structure factors have been deposited in the PDB with the accession codes 1H31, 1H32 and 1H33.

Magnetic Circular Dichroism of Hemoproteins.

Publisher Summary This chapter describes the progress made over the last 10 years in studies of the magnetic circular dichroism (MCD) spectra of heme models and proteins. The construction by Oxford Instruments of a split-coil superconducting magnet, in which the sample could be immersed in a liquid helium bath, necessitated the development of optical windows that remained strain free and capable of propagating circularly polarized light at 4.2 K. This led to the ability to make measurements routinely on samples of hemoproteins over the temperature range 1.5–300 K and up to fields of 6–7 T and opened the way for the measurement for the first time of MCD magnetization curves of metalloproteins and hence the methodology for the study of the ground-state magnetic properties of individual heme centers in proteins. It is now possible to measure the MCD spectra of hemoproteins in a solution of water and cryoprotectant mixtures over the wavelength range 195–5000 nm at magnetic fields up to 10 T, with good sample temperature control over the range 1.5–300 K. Although MCD studies in the vacuum ultraviolet region have been carried out using laboratory-based instruments and synchrotron sources, no reports have appeared of the spectra of hemoproteins in this wavelength region.

The Nitric Oxide Reductase Activity of Cytochrome c Nitrite Reductase from Escherichia coli

Cytochrome c nitrite reductase (NrfA) from Escherichia coli has a well established role in the respiratory reduction of nitrite to ammonium. More recently the observation that anaerobically grown E. coli nrf mutants were more sensitive to NO· than the parent strain led to the proposal that NrfA might also participate in NO· detoxification. Here we describe protein film voltammetry that presents a quantitative description of NrfA NO· reductase activity. NO· reduction is initiated at similar potentials to NrfA-catalyzed reduction of nitrite and hydroxylamine. All three activities are strongly inhibited by cyanide. Together these results suggest a common site for reduction of all three substrates as axial ligands to the lysine-coordinated NrfA heme rather than nonspecific NO· reduction at one of the four His-His coordinated hemes also present in each NrfA subunit. NO· reduction by NrfA is described by a Km of the order of 300 μm. The predicted turnover number of ∼840 NO· s–1 is much higher than that of the dedicated respiratory NO· reductases of denitrification and the flavorubredoxin and flavohemoglobin of E. coli that are also proposed to play roles in NO· detoxification. In considering the manner by which anaerobically growing E. coli might detoxify exogenously generated NO· encountered during invasion of a human host it appears that the periplasmically located NrfA should be effective in maintaining low NO· levels such that any NO· reaching the cytoplasm is efficiently removed by flavorubredoxin (Km ∼ 0.4 μm).

A model of the copper centres of nitrous oxide reductase (Pseudomonas stutzeri)

Nitrous oxide reductase (N2OR), Pseudomonas stutzeri, catalyses the 2 electron reduction of nitrous oxide to di‐nitrogen. The enzyme has 2 identical subunits (M 1 ∼ 70 000) of known amino acid sequence and contains ∼ 4 Cu ions per subunit. By measurement of the optical absorption, electron paramagnetic resonance (EPR) and low‐temperature magnetic circular dichroism (MCD) spectra of the oxidised state, a semi‐reduced form and the fully reduced state of the enzyme it is shown that the enzyme contains 2 distinct copper centres of which one is assigned to an electron‐transfer function, centre A, and the other to a catalytic site, centre Z. The latter is a binuclear copper centre with at least 1 cysteine ligand and cycles between oxidation levels Cu(II)/Cu(II) and Cu(II)/Cu(I) in the absence of substrate or inhibitors. The state Cu(II)/Cu(I) is enzymatically inactive. The MCD spectra provide evidence for a second form of centre Z, which may be enzymatically active, in the oxidised state of the enzyme. Centre A is structurally similar to that of CuA in bovine and bacterial cytochrome c oxidase and also contains copper ligated by cysteine. This centre may also be a binuclear copper complex.

The structure of Mycobacterium tuberculosis CYP125: Molecular basis for cholesterol binding in a P450 needed for host infection

We report characterization and the crystal structure of the Mycobacterium tuberculosis cytochrome P450 CYP125, a P450 implicated in metabolism of host cholesterol and essential for establishing infection in mice. CYP125 is purified in a high spin form and undergoes both type I and II spectral shifts with various azole drugs. The 1.4-Å structure of ligand-free CYP125 reveals a “letterbox” active site cavity of dimensions appropriate for entry of a polycyclic sterol. A mixture of hexa-coordinate and penta-coordinate states could be discerned, with water binding as the 6th heme-ligand linked to conformation of the I-helix Val267 residue. Structures in complex with androstenedione and the antitubercular drug econazole reveal that binding of hydrophobic ligands occurs within the active site cavity. Due to the funnel shape of the active site near the heme, neither approaches the heme iron. A model of the cholesterol CYP125 complex shows that the alkyl side chain extends toward the heme iron, predicting hydroxylation of cholesterol C27. The alkyl chain is in close contact to Val267, suggesting a substrate binding-induced low- to high-spin transition coupled to reorientation of the latter residue. Reconstitution of CYP125 activity with a redox partner system revealed exclusively cholesterol 27-hydroxylation, consistent with structure and modeling. This activity may enable catabolism of host cholesterol or generation of immunomodulatory compounds that enable persistence in the host. This study reveals structural and catalytic properties of a potential M. tuberculosis drug target enzyme, and the likely mode by which the host-derived substrate is bound and hydroxylated.

Characterization of active site structure in CYP121. A cytochrome P450 essential for viability of Mycobacterium tuberculosis H37Rv.

Mycobacterium tuberculosis (Mtb) cytochrome P450 gene CYP121 is shown to be essential for viability of the bacterium in vitro by gene knock-out with complementation. Production of CYP121 protein in Mtb cells is demonstrated. Minimum inhibitory concentration values for azole drugs against Mtb H37Rv were determined, the rank order of which correlated well with Kd values for their binding to CYP121. Solution-state spectroscopic, kinetic, and thermodynamic studies and crystal structure determination for a series of CYP121 active site mutants provide further insights into structure and biophysical features of the enzyme. Pro346 was shown to control heme cofactor conformation, whereas Arg386 is a critical determinant of heme potential, with an unprecedented 280-mV increase in heme iron redox potential in a R386L mutant. A homologous Mtb redox partner system was reconstituted and transported electrons faster to CYP121 R386L than to wild type CYP121. Heme potential was not perturbed in a F338H mutant, suggesting that a proposed P450 superfamily-wide role for the phylogenetically conserved phenylalanine in heme thermodynamic regulation is unlikely. Collectively, data point to an important cellular role for CYP121 and highlight its potential as a novel Mtb drug target.

Expression, purification and spectroscopic characterization of the cytochrome p450 cyp121 from mycobacterium tuberculosis

The CYP121 gene from the pathogenic bacterium Mycobacterium tuberculosis has been cloned and expressed in Escherichia coli, and the protein purified to homogeneity by ion exchange and hydrophobic interaction chromatography. The CYP121 gene encodes a cytochrome P450 enzyme (CYP121) that displays typical electronic absorption features for a member of this superfamily of hemoproteins (major Soret absorption band at 416.5 nm with alpha and beta bands at 565 and 538 nm, respectively, in the oxidized form) and which binds carbon monoxide to give the characteristic Soret band shift to 448 nm. Resonance Raman, EPR and MCD spectra show the protein to be predominantly low-spin and to have a typical cysteinate- and water-ligated b-type heme iron. CD spectra in the far UV region describe a mainly alpha helical conformation, but the visible CD spectrum shows a band of positive sign in the Soret region, distinct from spectra for other P450s recognized thus far. CYP121 binds very tightly to a range of azole antifungal drugs (e.g. clotrimazole, miconazole), suggesting that it may represent a novel target for these antibiotics in the M. tuberculosis pathogen.

Superoxide-mediated amplification of the oxygen-induced switch from [4Fe-4S] to [2Fe-2S] clusters in the transcriptional regulator FNR.

In Escherichia coli, the switch between aerobic and anaerobic metabolism is controlled primarily by FNR (regulator of fumarate and nitrate reduction), the protein that regulates the transcription of >100 genes in response to oxygen. Under oxygen-limiting conditions, FNR binds a [4Fe-4S]2+ cluster, generating a transcriptionally active dimeric form. Upon exposure to oxygen the cluster converts to a [2Fe-2S]2+ form, leading to dissociation of the protein into monomers, which are incapable of binding DNA with high affinity. The mechanism of cluster conversion together with the nature of the products of conversion is of considerable current interest. Here, we demonstrate that [4Fe-4S]2+ to [2Fe-2S]2+ cluster conversion, in both native and reconstituted [4Fe-4S] FNR, proceeds via a one electron oxidation of the cluster, to give a [3Fe-4S]1+ cluster intermediate, with the release of one Fe2+ ion and a superoxide ion. The cluster intermediate subsequently rearranges spontaneously to form the [2Fe-2S]2+ cluster, with the release of a Fe3+ ion and, as previously shown, two sulfide ions. Superoxide ion undergoes dismutation to hydrogen peroxide and oxygen. This mechanism, a one electron activation of the cluster, coupled to catalytic recycling of the resulting superoxide ion back to oxygen, provides a means of amplifying the sensitivity of [4Fe-4S] FNR to its signal molecule.