Peter Hildebrandt

Mutational Analysis of Deinococcus radiodurans Bacteriophytochrome Reveals Key Amino Acids Necessary for the Photochromicity and Proton Exchange Cycle of Phytochromes

The ability of phytochromes (Phy) to act as photointerconvertible light switches in plants and microorganisms depends on key interactions between the bilin chromophore and the apoprotein that promote bilin attachment and photointerconversion between the spectrally distinct red light-absorbing Pr conformer and far red light-absorbing Pfr conformer. Using structurally guided site-directed mutagenesis combined with several spectroscopic methods, we examined the roles of conserved amino acids within the bilin-binding domain of Deinococcus radiodurans bacteriophytochrome with respect to chromophore ligation and Pr/Pfr photoconversion. Incorporation of biliverdin IXα (BV), its structure in the Pr state, and its ability to photoisomerize to the first photocycle intermediate are insensitive to most single mutations, implying that these properties are robust with respect to small structural/electrostatic alterations in the binding pocket. In contrast, photoconversion to Pfr is highly sensitive to the chromophore environment. Many of the variants form spectrally bleached Meta-type intermediates in red light that do not relax to Pfr. Particularly important are Asp-207 and His-260, which are invariant within the Phy superfamily and participate in a unique hydrogen bond matrix involving the A, B, and C pyrrole ring nitrogens of BV and their associated pyrrole water. Resonance Raman spectroscopy demonstrates that substitutions of these residues disrupt the Pr to Pfr protonation cycle of BV with the chromophore locked in a deprotonated Meta-Rc-like photoconversion intermediate after red light irradiation. Collectively, the data show that a number of contacts contribute to the unique photochromicity of Phy-type photoreceptors. These include residues that fix the bilin in the pocket, coordinate the pyrrole water, and possibly promote the proton exchange cycle during photoconversion.

Light-induced Proton Release of Phytochrome Is Coupled to the Transient Deprotonation of the Tetrapyrrole Chromophore

The Pr → Pfr phototransformation of the bacteriophytochrome Agp1 from Agrobacterium tumefaciens and the structures of the biliverdin chromophore in the parent states and the cryogenically trapped intermediate Meta-RC were investigated with resonance Raman spectroscopy and flash photolysis. Strong similarities with the resonance Raman spectra of plant phytochrome A indicate that in Agp1 the methine bridge isomerization state of the chromophore is ZZZasa in Pr and ZZEssa in Pfr, with all pyrrole nitrogens being protonated. Photoexcitation of Pr is followed by (at least) three thermal relaxation components in the formation of Pfr with time constants of 230 μs and 3.1 and 260 ms. H2O/D2O exchange reveals kinetic isotope effects of 1.9, 2.6, and 1.3 for the respective transitions that are accompanied by changes of the amplitudes. The second and the third relaxation correspond to the formation and decay of Meta-RC, respectively. Resonance Raman measurements of Meta-RC indicate that the chromophore adopts a deprotonated ZZE configuration. Measurements with a pH indicator dye show that formation and decay of Meta-RC are associated with proton release and uptake, respectively. The stoichiometry of the proton release corresponds to one proton per photoconverted molecule. The coupling of transient chromophore deprotonation and proton release, which is likely to be an essential element in the Pr → Pfr photocon-version mechanism of phytochromes in general, may play a crucial role for the structural changes in the final step of the Pfr formation that switch between the active and the inactive state of the photoreceptor.

In Situ Spectroelectrochemical Investigation of Electrocatalytic Microbial Biofilms by Surface‐Enhanced Resonance Raman Spectroscopy

Metal-reducing bacteria not only play a key role in geochemical redox cycles, but also attract increasing attention in view of their relevance for microbial bioelectrochemical systems, a seminal sustainable technology. This growing research interest is triggered by the bacteria s capability to oxidize substrates such as acetate and to transfer the released electrons to an insoluble terminal electron acceptor, for example, iron-containing minerals in nature or a fuel cell anode in bioelectrochemical applications. The underlying electron-transfer (ET) mechanisms between the bacteria and the terminal electron acceptor may occur by different mechanisms, including direct and mediated electron transfer denoted as DET and MET, respectively. In the case of DET, the electrons are transferred from the respiratory chain in the cell to extracellular inorganic material via a complex architecture involving several outer membrane cytochromes (OMCs). These cytochromes are multiheme proteins whose function and number of heme groups may vary largely within the same family. Although several studies investigated the behavior of these proteins embedded in microbial biofilms of wild-type and mutant Geobacter sulfurreducens, the archetype bacteria family employing DET, the role of these cytochromes in the heterogeneous ET across the biofilm/electrode interface is far from clearly understood. This is particularly true since structural data are currently only known for two OMCs, namely, OmcF and OmcZ. 11] In this respect, spectroscopic techniques that can be applied to biofilms in situ may provide important structural information about the OMCs involved in the DET. To date, only two spectroscopic studies were devoted to the investigation of OMCs embedded in the cellular membrane. 13] The spectroscopic measurements of these works were carried out with washed and re-suspended cells, but did not refer to intact biofilms grown on an electrode. Herein, we present for the first time in situ spectroscopic characterization of OMCs in a catalytically active microbial biofilm. By measuring the electrochemical and spectroscopic properties of microbial cells embedded in their natural biofilm habitat, a more realistic picture on the natural electron transfer will be provided. Therefore, we have employed surface-enhanced resonance Raman (SERR) spectroscopy in combination with cyclic voltammetry (CV). SERR spectroscopy exploits the combination of the molecular resonance Raman (RR) and the surface-enhanced Raman (SER) effect to probe selectively the heme groups solely of the proteins in proximity of the electrode surface. 14] This powerful technique, in our case performed under strict electrochemical control, reveals the redox, coordination and spin states of the heme iron as well as the nature of its axial ligand, thereby providing important structural information that may complement the interpretation of electrochemical data obtained by CV. 16] The biofilms were grown at a constant potential on roughened (i.e. SER-active) silver electrodes using 10 mm acetate as substrate (see the Supporting Information for experimental details). These biofilms produced a maximum chronoamperometric current density of 600 mAcm 2 (Figure SI2 in the Supporting Information), which is in good agreement with previous studies using graphite anodes. The voltammetric behavior of the biofilms was monitored under turnover (Figure SI3) and nonturnover conditions [that is, with and without the substrate (e.g. acetate), respectively]. Figure 1 shows the CV behavior of such a biofilm for nonturnover conditions. The two redox couples that are proposed to be involved in the DET, Ef,1 and Ef,2, are centered at formal potentials of 282 mV and 363 mV, respectively (all potentials are reported versus the Ag/AgCl (3.0m KCl) reference electrode). The main overall shape and peak positions of the cyclic voltammogram shown in Figure 1 are very similar to those obtained on graphite electrodes in parallel experiments and in previous studies, showing that biofilm formation is not affected by the nature of the electrode material. The similarity between these CV traces and those obtained solely from biofilms of Geobacter sulfurreducens indicates that the biofilm is highly dominated [*] Dr. D. Millo, H. K. Ly, Prof. Dr. P. Hildebrandt Institut f r Chemie, Sekr. PC14, Technische Universit t Berlin Strasse des 17. Juni 135, 10623 Berlin (Germany) Fax: (+ 49)30-3142-1122 E-mail: diego.millo@tu-berlin.de

Spectroscopic Insights into the Oxygen-tolerant Membrane-associated [NiFe] Hydrogenase of Ralstonia eutropha H16

This study provides the first spectroscopic characterization of the membrane-bound oxygen-tolerant [NiFe] hydrogenase (MBH) from Ralstonia eutropha H16 in its natural environment, the cytoplasmic membrane. The H2-converting MBH is composed of a large subunit, harboring the [NiFe] active site, and a small subunit, capable in coordinating one [3Fe4S] and two [4Fe4S] clusters. The hydrogenase dimer is electronically connected to a membrane-integral cytochrome b. EPR and Fourier transform infrared spectroscopy revealed a strong similarity of the MBH active site with known [NiFe] centers from strictly anaerobic hydrogenases. Most redox states characteristic for anaerobic [NiFe] hydrogenases were identified except for one remarkable difference. The formation of the oxygen-inhibited Niu-A state was never observed. Furthermore, EPR data showed the presence of an additional paramagnetic center at high redox potential (+290 mV), which couples magnetically to the [3Fe4S] center and indicates a structural and/or redox modification at or near the proximal [4Fe4S] cluster. Additionally, significant differences regarding the magnetic coupling between the Nia-C state and [4Fe4S] clusters were observed in the reduced form of the MBH. The spectroscopic properties are discussed with regard to the unusual oxygen tolerance of this hydrogenase and in comparison with those of the solubilized, dimeric form of the MBH.

Highly conserved residues Asp-197 and His-250 in agp1 phytochrome control the proton affinity of the chromophore and Pfr formation

The mutants H250A and D197A of Agp1 phytochrome from Agrobacterium tumefaciens were prepared and investigated by different spectroscopic and biochemical methods. Asp-197 and His-250 are highly conserved amino acids and are part of the hydrogen-bonding network that involves the chromophore. Both substitutions cause a destabilization of the protonated chromophore in the Pr state as revealed by resonance Raman and UV-visible absorption spectroscopy. Titration experiments demonstrate a lowering of the pKa from 11.1 (wild type) to 8.8 in H250A and 7.2 in D197A. Photoconversion of the mutants does not lead to the Pfr state. H250A is arrested in a meta-Rc-like state in which the chromophore is deprotonated. For H250A and the wild-type protein, deprotonation of the chromophore in meta-Rc is coupled to the release of a proton to the external medium, whereas the subsequent proton re-uptake, linked to the formation of the Pfr state in the wild-type protein, is not observed for H250A. No transient proton exchange with the external medium occurs in D197A, suggesting that Asp-197 may be the proton release group. Both mutants do not undergo the photo-induced protein structural changes that in the wild-type protein are detectable by size exclusion chromatography. These conformational changes are, therefore, attributed to the meta-Rc → Pfr transition and most likely coupled to the transient proton re-uptake. The present results demonstrate that Asp-197 and His-250 are essential for stabilizing the protonated chromophore structure in the parent Pr state, which is required for the primary photochemical process, and for the complete photo-induced conversion to the Pfr state.

Redox and redox-coupled processes of heme proteins and enzymes at electrochemical interfaces

Modern bioelectrochemical methods rely upon the immobilisation of redox proteins and enzymes on electrodes coated with biocompatible materials to prevent denaturation. However, even when protein denaturation is effectively avoided, heterogeneous protein electron transfer is often coupled to non-Faradaic processes like reorientation, conformational transitions or acid-base equilibria. Disentangling these processes requires methods capable of probing simultaneously the structure and reaction dynamics of the adsorbed species. Here we provide an overview of the recent developments in Raman and infrared surface-enhanced spectroelectrochemical techniques applied to the study of soluble and membrane bound redox heme proteins and enzymes. Possible biological implications of the findings are critically discussed.

Why Does the Active Form of Galactose Oxidase Possess a Diamagnetic Ground State

The relative orientation of the two magnetic orbitals, the CuII d x 2-y 2 orbital and the half-occupied π orbital of the tyrosyl radical, is the key to answering the question in the title. The arrangement shown (CuII -O-C bond angle of about 130° and a dihedral angle of about 90° between the x,y plane of the CuII polyhedron and the tyrosyl ring plane) leads to an overlap of the orbitals, which results in a singlet ground state.

Phenoxyl radical complexes of chromium(III), manganese(III), cobalt(III), and nickel(II)

Abstract The tetradentate monoanionic macrocycles 1,4-dimethyl-7-(3-tert-butyl-5-methoxy-2-hydroxybenzyl)-1,4,7-triazacyclononane, H(L1), and 1,4-di-iso-propyl-7-(3,5-di-tert-butyl-2-hydroxybenzyl)-1,4,7-triazacyclononane, H(L2), form in the presence of a bidentate coligand 1,3-diphenyl-1,3-propanedionate, Ph2acac, 1,3-dimethyl-1,3-propanedionate, acac, or 1,3-di-tert-butyl-1,3-dionate, Bu2acac−, very stable octahedral complexes with di- or trivalent metal ions: [MIII(L1)(Ph2acac)](ClO4) M=CoIII (1), CrIII (3), MnIII (7), GaIII (11); [MII(L1)(Ph2acac)] M=NiII (9); [MIII(L2)(Bu2acac)](ClO4) M=CoIII (2), CrIII (4), MnIII (8), GaIII (12), [MII(L1)(Bu2acac)] M=NiII (10); [CrIII(L2)(acac)](ClO4) (5), [CrIII(L2)(C2O4)] (6). All of these monophenolatometal complexes can be electrochemically, reversibly one-electron oxidized yielding stable phenoxyl radical complexes in solution. Electronic, resonance Raman and EPR spectra prove that the phenoxyl ligand is coordinated to the corresponding metal ion. The electronic ground states of complexes containing a paramagnetic transition metal ion with dn electron configuration and a bound phenoxyl radical (S=1/2) are attained via an intramolecular anti- or ferromagnetic exchange coupling mechanism the nature of which depends on the actual dn configuration of the central metal ion: a half-filled t2g subshell allows antiferromagnetic coupling, for example [3] 2+, [4] 2+, [5] 2+ and [6] + have an St=1 ground state. In contrast, unpaired electrons in an eg subshell enforce a ferromagnetic coupling: [9] 2+, [10] 2+ have an St=3/2 ground state. The crystal structures of 1, 5, and 9 are reported.

Phenoxyl-copper(II) complexes: models for the active site of galactose oxidase

Abstract The reaction of the macrocycles 1,4,7-tris (3,5-di-tert-butyl-2-hydroxy-benzyl)-1,4,7-triazacyclononane, L1H3, or 1,4,7-tris(3-tert-butyl-5-methoxy-2-hydroxy-benzyl)-1,4,7-triazacyclononane, L2H3, with Cu(ClO4)2·6H2O in methanol (in the presence of Et3N) affords the green complexes [CuII(L1H)] (1), [CuII(L2H)]·CH3OH (2) and (in the presence of HClO4) [CuII(L1H2)](ClO4) (3) and [CuII(L2H2)] (ClO4) (4). The CuII ions in these complexes are five-coordinate (square-base pyramidal), and each contains a dangling, uncoordinated pendent arm (phenol). Complexes 1 and 2 contain two equatorially coordinated phenolato ligands, whereas in 3 and 4 one of these is protonated, affording a coordinated phenol. Electrochemically, these complexes can be oxidized by one electron, generating the phenoxyl-copper(II) species [CuII(L1H)]+ ·, [Cu(L2H)]+ ·, [CuII(L1H2)]2+ ·, and [CuII(L2H2)]2+ ·, all of which are EPR-silent. These species are excellent models for the active form of the enzyme galactose oxidase (GO). Their spectroscopic features (UV-VIS, resonance Raman) are very similar to those reported for GO and unambiguously show that the complexes are phenoxyl-copper(II) rather than phenolato-copper(III) species.

Lewis Acid Trapping of an Elusive Copper–Tosylnitrene Intermediate Using Scandium Triflate

High-valent copper-nitrene intermediates have long been proposed to play a role in copper-catalyzed aziridination and amination reactions. However, such intermediates have eluded detection for decades, preventing the unambiguous assignments of mechanisms. Moreover, the electronic structure of the proposed copper-nitrene intermediates has also been controversially discussed in the literature. These mechanistic questions and controversy have provided tremendous motivation to probe the accessibility and reactivity of Cu(III)-NR/Cu(II)N(•)R species. In this paper, we report a breakthrough in this field that was achieved by trapping a transient copper-tosylnitrene species, 3-Sc, in the presence of scandium triflate. The sufficient stability of 3-Sc at -90 °C enabled its characterization with optical, resonance Raman, NMR, and X-ray absorption near-edge spectroscopies, which helped to establish its electronic structure as Cu(II)N(•)Ts (Ts = tosyl group) and not Cu(III)NTs. 3-Sc can initiate tosylamination of cyclohexane, thereby suggesting Cu(II)N(•)Ts cores as viable reactants in oxidation catalysis.