Nitrile Hydratase from Rhodococcus rhodochrous J1 Contains a Non-Corrin Cobalt Ion with Two Sulfur Ligands

Abstract

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 ...

H2O2/Nitrite‐Induced Post‐translational Modifications of Human Hemoglobin Determined by Mass Spectrometry: Redox Regulation of Tyrosine Nitration and 3‐Nitrotyrosine Reduction by Antioxidants

“…Interestingly, the cobalt-containing cyanocobalamin is very efficient in catalyzing the reduction of 3NT, presumably by a mechanism similar to the reduction of the Co 3 + ion to Co 2 + by thiol-containing antioxidants. [39] These results suggest that protein tyrosine nitration in vivo is a redox-regulated process. Many important signaling cascades are regulated by redox states.…”

Self-subunit Swapping Chaperone Needed for the Maturation of Multimeric Metalloenzyme Nitrile Hydratase by a Subunit Exchange Mechanism Also Carries Out the Oxidation of the Metal Ligand Cysteine Residues and Insertion of Cobalt

“…N-771 in the presence of ferric citrate in vitro has been observed (34). Cobalt exists as a non-corrin low-spin Co 3ϩ ion in H-NHase in R. rhodochrous J1 (40) and NHase in P. putida NRRL-18668 (7), suggesting that the holo-␣-subunit of L-NHase (holo-␣ 2 ␤ 2 ) and the holo-␣-subunit of holo-␣e 2 (which is the source of that of holo-␣ 2 ␤ 2 ) should also have a non-corrin low-spin Co 3ϩ ion. Therefore, the post-translational oxidation of the cysteine residues was associated with the oxidation of Co 2ϩ to Co 3ϩ , because Co 2ϩ was used as the cobalt donor in the in vitro experiments.…”

Structure of the Photoreactive Iron Center of the Nitrile Hydratase from Rhodococcus sp. N-771

“…It is widely believed that the metal site structure of the cobalt-containing NHases is closely related with that of the iron-containing one. Indeed, recent ESR and extended x-ray absorption fine structure studies showed that the metal site of the NHase from Rhodococcus rhodochrous J1 was a mononuclear low-spin six-coordinate Co 3ϩ ion with the ligand field of mixed sulfur, and nitrogen or oxygen donor atoms (4).…”