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The influence of ligand structure on Ni(III)/Ni(II) redox potentials is examined in order to elucidate those factors which lead to unusually low values for this couple, such as are found in (NiFe)‐hydrogenases.

A purification procedure for the periplasmic hydrogenase from Desulfovibrio vulgaris (Hildenborough, Na- MATERIALS AND METHODSGrowth of Organisms and Preparation of Crude Extract. 57Fe-labeled D. vulgaris (Hildenborough, NCIB 8303) was grown for 38 hr in lactate/sulfate medium containing the following components per liter: sodium lactate (60%), 12.5 ml; NH4Cl, 2 g; MgSO4-7H2O, 2 g; K2HPO4, 0.5 g; Na2SO4, 4 g; CaCl2 2H2O, 0.035 g; Na2S-9H20, 0.25 g; 57Fe (enrichment, 95%), 1 mg; EDTA, 2 mg; cysteine hydrochloride, 0.125 g; mineral solution (9), 1 ml. The pH of the medium was 7.2 after autoclaving. Cells (407 g) from 200 liters were harvested and then suspended in 400 ml of 10 mM Tris-HCl (pH 7.6) and frozen at -80°C for 2 days. The cells were then slowly defrosted for about 20 hr and were centrifuged at 19,000 x g for 45 min. The supernatant was called crude extract.Purification of Hydrogenase. All purification procedures were carried out in air, at 40C, and the pH of the buffers was 7.6 (measured at 50C). A summary of the results of a typical purification is presented in Table 1.First DEAE-Bio-Gel column. The crude extract was placed on a DEAE Bio-Gel column (6 x 32.5 cm) and washed with 500 ml of 0.01 M Tris-HCl buffer, and the proteins were eluted with two Tris-HCl linear gradients ( Hydroxylapatite column. The collected hydrogenase-containing fractions were loaded onto a hydroxylapatite (BioRad) column (4.5 x 24.5 cm) and the column was washed with 100 ml of 1 mM potassium phosphate (KP) buffer at a flow rate of 40 ml per hour. The protein was then eluted by two phosphate linear gradients (1.25 liters of 1 mM KP buffer and 1.25 liters of 0.2 M KP buffer, 625 ml of 0.2 M KP buffer and 625 ml of 0.3 M KP buffer). The hydrogenase activity was detected between 1650 ml and 2390 ml and the volume concentrated to 15 ml by using a diaflow apparatus with a YM 30 membrane.Second DEAE-Bio-Gel column. The concentrated protein solution was diluted 1:10 with 10 mM Tris-HCl and then applied to the second DEAE-Bio-Gel column (4.5 x 22.5 cm). The column was washed with 200 ml of 0.01 M Tris-HCl, and then a linear gradient was constructed (750 ml of 0.01 M Tris-HCl and 750 ml of 0.2 M Tris-HCI). The hydrogenase was collected between 1200 and 1330 ml and the volume was concentrated to 14 ml.Sephacryl S-200 column. The protein was loaded on a Sephacryl S-200 column (5.4 x 85 cm) and eluted with 0.05 M KP buffer at a flow rate of 20 ml/hr. The protein was collected in 5 ml-fractions between 650 and 700 ml. Purity of the hydrogenase was established by polyacrylamide disc electrophoresis (10) as well as NaDodSO4/acrylamide electrophoresis (11). The purified hydrogenase had a specific activity of 4800 ,umol of H2/min per mg of protein and an A400 nm/A280 nm ratio of 0.36.Assays and Metal Determination. Hydrogenase activity was determined by the H2 evolution assay (12). Hydrogen was determined by means of a Varian 4600 gas chromatograph (4) and protein, by the Bradford method (13) using bovine serum albumin as a reference st...

oxygen molecule on the iron(iii) catecholate moiety.

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COMMUNICATIONS[(I] Scveriil comp~)tind\ of thc type PhSeSiR, (R, = Me,, Et,. Me,Ph. Me,rBu. Ph,iBu) Mere Acreened for this purpose; however, compound 1 was hest suited fool the radicnl chain group transfer reaction because ofits stability to hydrolysis [J] 1351. _I. H. Byen. T G. Gleason, K S. Knight. J. Clirm. So<,. C/iriii. Cmiiiiuii. 19YI. 354 356. 1x1 T. H:iinnd,i. A Nishida. 0. Yonemitsu. J Ain. Cheni So< 1986. 108, 140-145: 1' . Hamada. .A Nishida. 0 Yonemitsu. ihid. 1980. /02. 3978-3Y80. [O] 1). Rchin. A Wcllei.. 1 ,~. J C/im. 1970. 8. 259 [7] M. Nchcomb. D J. Marquardt. M. U. Kumar. 7?7rtro/itvhii 19Y0, 46. 2345 [lo] The ouidatioii potential (€(";I = 1.2X eV;SCE) and energy orexcitation (E,,<> = 367 37 k J ) value\ for DMN wcre taken from ref. [8]. whereas the E\T',"]of 1 wiis rmined by cyclic voltammetry. by using ii PAR 173 potentiostat coupled with a PAR 175 universal progammer, and the data werc recorded on :I RE0091 X-Y recorder: Pt UME (Ultra micro electrode) working electrode. Pt foil counter electrode; tetrabutylammonium hexafluurohorate ITBABF,) in dry CH ,CN as supporting electrolyte. The CH,CN rolution wi\ degassed hy argon bubbling for 10 min before measurement. The sciiii rille wii\ 500 iiiVb-' and values :ire relative to SCE. [12] TIIS G i b b frcc cnergy was determined with theequation AG,, = E[,r;l -Ei';d'. i n ~. h i c h the El'';] value of H,A estimated by cyclic \oltanimetry [lo] w a y 1.OX4cV'SCI~ and the E\'Sdl value for DMN" was 1.28 eV:SCE [XI.

Dedicated to Professor Christoph Elschenbroich on the occasion of his 70th birthday Spin crossover and valence tautomerism are examples of processes that can be utilized as a basis for achieving molecular switches. [1] Whereas the spin-crossover process is characterized by a temperature-, pressure-, or light-induced change of the electronic state of the metal ion to one with a different spin multiplicity, [2] valence tautomerism entails an intramolecular redox reaction between a metal ion and a coordinated ligand, which, in a few instances, is accompanied by a change in the spin state of the metal ion. [3] Various reported low-spin cobalt(III) catecholate complexes, which can be transformed into high-spin cobalt(II) semiquinonate complexes by raising the temperature, provide excellent examples of the latter process. In contrast, spin-crossover chemistry is dominated by octahedral iron(II) complexes with a FeN 6 coordination sphere; [2] however, there are only very few known octahedral cobalt(II)-containing spin-crossover complexes. [4] Herein we describe the first cobalt(II) semiquinonate complex that displays spin-crossover properties rather than valence tautomerism.The starting point of our investigation was the olive-green cobalt(III) 3,5-di-tert-butylcatecholate (dbc 2À ) complex Me 2 )(dbc)](BPh 4 )·0.8 MeCN·0.2 Et 2 O (1) containing the dimethyl derivative of the tetraazamacrocyclic ligand 2,11diaza[3.3](2,6)pyridinophane (L-N 4 Me 2 ) as coligand. This complex was obtained in 42 % yield by oxidation of the red cobalt(II) catecholate complex Me 2 )(dbc)] (prepared in situ from equimolar solutions of cobalt(II) perchlorate, L-N 4 Me 2 , and 3,5-di-tert-butylcatecholate) with ferrocenium tetrafluoroborate ([Fe(Cp) 2 ](BF 4 ); Cp = cyclopentadienyl), followed by a metathesis reaction with sodium tetraphenylborate (Scheme 1). In accordance with the description of 1 as a cobalt(III) catecholate complex, solutions and solids of this substance are diamagnetic. X-ray structure analysis of 1 also supports this assignment. [6] Figure 1 shows a perspective view of the complex cation in 1. Because of the small size of the macrocyclic ring, the coordinated ligand L-N 4 Me 2 is folded along the N amine -N amine axis, thereby rendering a distorted cis-octahedral coordina-Scheme 1. Preparation of compounds 1 and 2. Figure 1. Perspective view of the complex cation in 1 showing 50 % thermal ellipsoids; selected bond lengths []: