29
Chapter 4
Experimental Details and Methods for Evaluation of Results
In this work the hydrogenase from D. vulgaris Miyazaki F has been investigated by means of electron paramagnetic resonance spectroscopy in its various redox states. This chapter describes the applied biochemical methods for sample preparation, experimental realization of the magnetic resonance tech-niques, as well as the methods and procedures for the data processing of the experimental data and subsequent interpretation of the spectra. Single crystals of the enzyme have been examined by orien-tation dependent cw-EPR spectroscopy. The results are presented in Chapter 5. 61Ni isotope labelled hydrogenase from D. vulgaris Miyazaki F has been prepared. The observed hyperfine couplings led to valuable information about spin density distribution, which is discussed in Chapter 6. In Chapter 7 the results of pulsed EPR, as well as double resonance experiments on the oxidized (Ni-A/B), reduced (Ni-C) and the light induced (Ni-L) states of the hydrogenase from D. vulgaris Miyazaki F are pre-sented. They revealed hyperfine couplings of the hydrogen and nitrogen nuclei in the vicinity of the spin-carrying center, which provided important information about the geometry of the active site.
4.1 Sample Preparation
4.1.1 Chemicals, Biochemicals and Materials
HEPES (2-[4-(2-hydroxyethyl)-piperazinyl]-ethane-sulfonic acid), Tris (tris-(hydroxymethyl)-aminomethan), HCl 37%, NaOH, (p.A.), Merck, Darmstadt, Germany
Sephadex G-50, Pharmacia, Freiburg, Germany; Microcon YM-10 microconcentrator, Millipore, Schwalbach, Germany
MPD (2-methyl-2,4-pentane-diol) ( 99%), Fluka, Neu-Ulm, Germany methyl viologen, Fluka, Neu-Ulm, Germany
30 4.1 Sample Preparation D2O (99.9 atom %), Isotec Inc., A. Matheson Tri-Gas Company; H172 O (40 atom %),61Ni metal (92%), Campro Scientific, Veenendaal, Netherlands
H2(5.0), 1% H2in 99% He (5.0), Ar (5.0), D2(2.7), Messer-Griesheim, Berlin, Germany; catalyst for gas purification: R3-11G, BASF, Ludwigshafen, Germany
The membrane-bound hydrogenase from D. vulgaris Miyazaki F isolated and purified as de-scribed previously (enzyme solutions [62] and single crystals [112]) was kindly provided by Y.
Higuchi and H. Ogata (Kyoto, Japan) and later by B. Katterle (M¨ulheim).
4.1.2 Oxidized States
Samples at different pH values were prepared by gel filtration over a Sephadex G-50 column equili-brated with the respective buffer, according to Penevsky [113] and successive reconcentration of the eluate with a microconcentrator. Concentrations lower than 5-10 mM were stable for several hours or days. At higher concentrations the enzyme denatured notably. For pH 5.5-6.5 HEPES, 25-100mM and for pH 7.0-8.0 Tris/HCl, 25-100mM was used. However, if nothing indicated as experimental con-ditions, then the samples were prepared in 25 mM Tris/HCl at pH 7.4. The enzyme concentration was determined according to the method described by Bradford [114]. The (systematic) error of the protein determination method is estimated to be about 10 %.
Single crystals of the hydrogenase were grown by the sitting drop vapor diffusion method from 33% MPD (2-methyl-2,4-pentane-diol) and 25 mM Tris/HCl buffer solution at pH 7.4 [112]. The crys-tals belonged to the orthorhombic space group P212121 and contained four magnetically inequivalent molecules (sites) per unit cell. Their respective orientations are related to each other by a 21screw axis.
Isotope Exchange Experiments Solvent exchange with the respective buffer in D2O or H172 O was obtained by repeated concentration of the enzyme solution with a microconcentrator to about 1/20 of the initial volume and redilution with the respective buffer solution for at least 6 times. Tris buffer of the isotope exchanged solvent was prepared by thorough lyophyllization of 1 ml portions of the respective buffer solution in H2O and redilution in the solvent. To achieve 25mM Tris/DCl pDtrue 7.4, a dried solution of Tris/HCl at pH 7.1 was used [12, 115] and for the H172 O buffer pH 7.4. Upon the H/D isotope exchange procedure, the enzyme behaved more sensible, it denatured faster than in respective procedure in H2O.
The reductions of D2O exchanged samples were undertaken under 100% D2 gas, following the procedure described in section 4.1.3.
For the61Ni labeling, bacteria were grown in a 100 l steel fermenter according to the protocol given in [62] with the addition about 150 mg of61NiCl2. The 61NiCl2 salt was prepared by dissolving 61Ni metal in HCl 1:1 diluted in H2O at 70-90 C for 13h. The resulting clear green solution was evaporated.
The green precipitate (NiCl26H2O) was dried at 110 C until its weight did not decrease any more. A yellow powder (NiCl2) with a yield of 98 % was obtained.
Experimental Details and Methods for Evaluation of Results 31 Preparation of the Ni-A and Ni-B states In the as isolated samples of D. vulgaris Miyazaki F EPR signals of both species, Ni-A and Ni-B, are detectable. Depending on the respective enzyme prepara-tion, the ratio of Ni-A:Ni-B was 3:7 - 1:1. In order to obtain pure Ni-B, the samples were reduced as described in section 4.1.3. The reoxidation to Ni-B to relative spin concentrations of about or more 90%
Ni-B with respect to Ni-A was carried out by reoxidation of the sample, cooled in an ice-bath, with air for about 10-30 minutes. The time of exposure to air depended on the enzyme concentration. Upon longer incubation times signals at g 2.26, 2.20, 2.14 emerged. The percentage of Ni-A in the EPR spectra could be increased by reoxidizing the sample under argon atmosphere at room temperature.
However, only up to 60 % Ni-A was obtained with satisfying spin concentrations.
4.1.3 Reduced States
Preparation of the Ni-C State in Solutions and in Single Crystals All experiments under exclusion of oxygen have been undertaken using a home-built gassing apparatus as described by Beinert et al.
[116].
The enzyme samples were activated and reduced under pure hydrogen gas or deuterium gas at 37 C and atmospheric pressure for 2-3 h (3-6 h with D2 gas) directly in the quartz EPR tubes. In order to gain higher spin concentrations of Ni-C, the active enzyme was treated with a mixture of 1% H2in 99%
He for about 30 minutes. To ensure a homogeneous distribution of the gas in the solution, the samples were stirred with small stirring bars (1 x 5-10 mm). The activated samples were then rapidly frozen in liquid nitrogen.
In order to gain the ’unsplit’ form of the reduced state, i.e. the reduced form of the active site with oxidized and thus diamagnetic [4Fe-4S]-clusters, the H2reduced sample was equilibrated under Ar at low temperatures (ice-bath) in the EPR tube and mixed with a small stirring bar until the splitting of the EPR transitions was diminished. A reasonable diminution was usually achieved with incubation times of about 15-30 minutes.
The size of the crystals used for EPR experiments was approximately 1-2 mm x 0.5 mm x 0.5 mm.
The crystals had to be soaked with a precipitant solution containing 1-2 mM methyl viologen for at least 10-14 days (Incubation times longer than 6 weeks deactivated the crystallized enzyme completely).
Then the crystals were placed in thin walled quartz capillaries and the precipitant solution was removed until the crystal was only slightly covered by the liquid. The capillaries were marked to indicate the initial position for the angular dependent EPR experiments and then transferred to quartz EPR tubes.
The crystals were then reduced under pure hydrogen at 37 C for several hours. The success of reduction of single crystal samples was checked by EPR spectroscopy at room temperature. The samples were then rapidly frozen in liquid nitrogen.
Conversion to Ni-L in Solutions and in Single Crystals Frozen samples in the Ni-C state were illu-minated with white light at low temperatures either in a liquid nitrogen bath or directly in the resonator
32 4.2 Magnetic Resonance Spectroscopy Setup