No abstract

The iron K-edge X-ray absorption spectrum of Rhodococcus sp. R312 (formerly Brevibacterium sp. R312) nitrile hydratase in frozen solutions at pH 7 and 9 has been analyzed to determine details of the iron coordination. EXAFS analysis implies two or three sulfur ligands per iron and overall six coordination; together with previous EPR and ENDOR results, this implies an N3S2O ligation sphere. The bond lengths from EXAFS analysis [rav(Fe-S) = 2.21 A at pH 7.3; rav(Fe-N/O) = 1.99 A] support cis coordination of two cysteine ligands and conclusively rule out nitric oxide coordination to the iron, a possibility proposed on the basis of an FTIR difference experiment [Noguchi, T., Honda, J., Nagamune, T., Sasabe, H., Inoue, Y., & Endo, I. (1995) FEBS Lett. 358, 9-12]. The higher-frequency EXAFS can be simulated well by inclusion of multiple scattering from two or three imidazole ligands; the fit to the data is improved if first-sphere multiple scattering pathways are also included. A slight shortening (by 0.02 +/- 0.01 A) of one or both Fe-S bonds when the pH is raised from 7.3 to 9.0 is consistent with shifts observed in the Raman spectrum [Brennan et al. (1996) Biochemistry 35, 10068-10077].

We present spectroscopic data that show that a nitrile hydratase from Rhodococcus rhodochrous J1 is the first reported example of a native protein that contains a non-corrin Co 3+ ion with a mixed S and N(O) ligand field. Nitrile hydratases catalyze the addition of water to nitriles, yielding amides as the exclusive product, 3 and are used as industrial catalysts for the production of acrylamide. 4 The most thoroughly characterized nitrile hydratase is from Rhodococcus sp. R312 5 and is a (R ) 2 tetramer that contains two low-spin non-heme ferric ions of unknown function. These metal ions exist in a tetragonally distorted octahedral ligand field of three histidine imidazoles, two cysteine thiolates, and a hydroxide. 6-8 Two cobaltcontaining nitrile hydratases have been identified in R. rhodochrous J1. 9 We purified one of those enzymes 10,11 a multimer of R heterodimers totaling approximately 500 000 Da and containing non-corrin Co 3+ . 10 We measured the cobalt 12 and the protein 13 concentrations of samples of purified enzyme and found one cobalt ion per (R ). The same experiment yielded an unusually high 280 (2.7 (mg/mL) -1 cm -1 ), consistent with an earlier report. 10 EPR spectra of concentrated samples (0.3 mM cobalt) showed no signals attributable to the protein from 4 to 77 K, consistent with the presence of Co 3+ . When treated with sodium dithionite and methyl viologen, the samples developed an EPR spectrum characteristic of low-spin Co 2+ (Figure 1; g 1,2,3 ) 2.378, 2.206, 1.998; A Co 1,2,3 ) 58, 11, 97 G). 14 The cobalt K-edge X-ray absorbance spectrum of this nitrile hydratase (in the presumed Co 3+ form) is very similar to the Fe 3+ K-edge spectrum of the Rhodococcus sp. R312 enzyme 15,16 (Figure 2a,b). Using the method of Roe et al., 17 the area (in units of eV (% edge height)) of the lowest energy pre-edge peak in the cobalt spectrum (assigned to a 1s f 3d transition) is 6.3, slightly larger than areas we obtained for six-coordinate Co-(S 2 CNEt 2 ) 3 (3.6), Co(acac) 3 (3.9), and [Co(en) 3 ]Cl 3 (4.6), but much smaller than that found for the four-coordinate Co(im) 2 -Cl 2 (16.8). The size of the pre-edge peak is consistent with six-or possibly five-coordinate cobalt in nitrile hydratase, with distortions from octahedral symmetry that increase the peak area by approximately 50% compared to those of symmetrical sixcoordinate models. The pre-edge peak for the six-coordinate 7,8,16 iron in Rhodococcus sp. R312 nitrile hydratase is also approximately 50% larger than those of symmetrical six-coordinate Fe 3+ model complexes. 16 The best fits of the first sphere Fourier-filtered EXAFS are shown in Figure 2c and assume two sulfur scatterers at 2.20 Å and three ( ν 2 ) 1.3) 18 or four ( ν 2 ) 1.5) nitrogen scatterers at 1.95 Å. Any other integer values of n S or n N in two-shell fits gave ν 2 g 3.0 and were rejected on the basis of the criterion that ν 2 for a correct model is expected to be within one unit of the minimum ν 2 obtained. 19 The value of ν 2 increased by only 0.2-0.3 for each nitrogen ...

No abstract

No abstract

rangements of phosphine ligands and require passage through several intermediate TTP topologies in order to return to the ground-state arrangement with two eclipsed prismatic phosphines and a capping phosphine on the opposite prismatic face. Conclusions13C and 31P CP/MAS NMR data obtained over a range of temperatures are consistent with a tricapped-trigonal-prismatic structure for W(PMe3)3H6, with two phosphine ligands in eclipsed prismatic sites and the third in the opposite capping site. In contrast to the results of previous crystallographic studies on such compounds, the NMR spectra suggest that the two prismatic phosphine ligands in each molecule are slightly inequivalent. At temperatures above ambient, interchange of ligand functionality for the phosphine ligands is observed by magnetization-transfer experiments and, at still higher temperatures, by simulation of the exchange-broadened NMR line shapes observed experimen-tally. Rate data from the two methods of analysis suggest Arrhenius activation parameters for ligand functionality interchange of £a = 148.8 ± 15 kJ mol'1 and A -6.6 X 1023 s'1. The rate of functionality interchange reaches ca. 2000 Hz by the decomposition point of the material (381 K). A mechanism for this exchange has been proposed, involving the "double rearrangement" TTP(ground state) -MSA ^TTP(excited state) **=* MSA 5=* TTP(ground state)In this mechanism slight stretches of the polytypal edges result in interchange of ligand functionality without the need for unfavorable spatial permutation of the phosphine ligands.Acknowledgment. J. Pound is thanked for a sample of W-(PMe3)3H6, and Drs.

Tyrosine hydroxylase (TH) was purified from tumours of rat phaeochromocytoma (PC12) cells by a three-step purification procedure giving 30 mg of pure enzyme in 3 days. The enzyme sedimented with an S(eo),w value of 9.2 S and revealed an apparent subunit molecular mass of 62 kDa with a minor 60 kDa component. Two-dimensional gel isoelectric focusing/electrophoresis and tryptic digestion revealed that the heterogeneity could be accounted for by limited proteolysis of the 62 kDa component and the presence of covalently bound phosphate. The enzyme had a strong blue-green colour (epsilon 700 = 3.1 +/- 0.2 mM-iron-1.cm-1). The resonance Raman spectrum obtained with lambda excitation = 605 nm revealed the presence of an Fe(III)-catecholamine complex in the isolate enzyme, similar to that observed in the bovine adrenal enzyme [Andersson, Cox, Que, Flatmark & Haavik (1988) J. Biol. Chem. 263, 18621-18626]. In the rat PC12 enzyme, all of the iron present (0.53 +/- 0.03 atom per subunit) seems to be chelated by the feedback inhibitors (0.49 +/- 0.05 mol of dopamine and 0.10 +/- 0.03 mol of noradrenaline per mol of subunit). The e.p.r. spectra at 3.6 K show g-values at 7.0, 5.2 and 1.9 as observed for other catecholate-complexed enzymes. After phosphorylation of serine-40 and addition of L-tyrosine a new rhombic (magnitude of E/D = 0.33) e.p.r. species could be observed. Phosphorylation of serine-40 by cyclic AMP-dependent protein kinase increased the catalytic activity; depending on assay conditions, up to 80-110-fold activation could be observed when measured at high TH (i.e. high endogenous catecholamine) concentration.

No abstract