The structure of a derivative of coenzyme F430 from methanogenic bacteria, the bromide salt of 12,13-diepi-F430 pentamethyl ester (5, X = Br), was determined by X-ray structure analysis. It reveals a more pronounced saddle-shaped out-of-plane deformation of the macrocycle than any hydroporphinoid Ni complex investigated so far. The crystal structure confirms the constitution proposed for coenzyme F430 (2) and shows that in the epimer 5, the three stereogenic centers in ring D, C(17), C(18), and C(19), have the (17s)-, (lXS)-, and (19R)-configuration, respectively. Deuteration and 2D-NMR studies independently demonstrate that native coenzyme F430 (2) has the same configuration in ring D as the epimer 5. Therefore, our original tentative assignment of configuration at C(19) and C(18) [l] has to be reversed. This completes the assignment of configuration for all stereogenic centers in coenzyme F430, which has the structure shown in Formula 2.1. Introduction. -In the previous papers in this series [1-4], we reported on the structure elucidation of coenzyme F430 [5] [6], a hydroporphinoid nickel complex which plays a central role in the energy metabolism of methanogenic bacteria [7]. As a component of methyl-coenzyme M reductase, it catalyzes the reductive cleavage of S-methylcoenzyme M to coenzyme M and methane [8] [9].Our structural studies which were carried out in close collaboration with the group of R . K. Thauer at the University of Marburg led to the structural formula 1 for coenzyme F430 (R = H). The constitution was determined largely by NMR analysis of the pentamethyl ester F430M in combination with a series of biosynthetic incorporation experiments using specifically I3C-labeled precursors [la] and UV/VIS studies of synthetic hydrocorphinoid model complexes [ 1 b]. Making extensive use of 1 D-NOE-difference spectroscopy, it was possible to establish the relative configuration at the six stereogenic centers in rings A and B and the trans-arrangement of the ring-C side chains, but not the configurational relationship between rings B and C. The absolute configuration at C( 12) and C( 13) was deduced by ozonolytic excision of ring C and CD-spectroscopic correlation of the resulting succinimide derivative with reference compounds of known absolute configuration [la]. The absolute configuration of the A/B fragment followed from comparison of the CD spectra of nickel(I1) isobacteriochlorinate derivatives prepared from F430 with the CD spectrum of nickel(I1) sirohydrochlorinate octamethyl ester [4].

The reduction of methyl-coenzyme M (CH3SCoM) to methane in methanogenic bacteria is dependent on component B (N-7-mercaptoheptanoyl-O-phospho-L-threonine, HSHTP). We report here that S-methylcomponent B (N-7-(methylthio)heptanoyl-O-phospho-L-threonine, CH3SHTP) can substitute for neither CH3SCoM nor HSHTP in the methyl-CoM reductase reaction. Rather, CH3SHTP proved to be an inhibitor competitive with HSHTP (apparent Ki = 6 gM) and noncompetitive with CH3SCoM. These results make it very unlikely that HSHTP functions as a methyl group carrier. A role for HSHTP as direct electron donor for CH3SCoM reduction to CH4 is proposed.

(18)) sowie die trans-Anordnung der beiden Substituenten am Ring C, beruht auf den Ergebnissen einer ' H-NMR-NOE-differenzspektroskopischen Analyse von F430M. Infolge der trigonalen Struktur des meso-C-Atoms C(10) zwischen den Ringen B und C war es mit der NOE-Methodik nicht moglich gewesen, die Strukturbereiche der Ringe B und C konjigurativ zu korrelieren. Dies ist der Grund dafur, dass ein experimenteller Beweis der ahsoluten Konfiguration von F430 bislang fehlte, wiewohl es moglich war, durch ozonolytische Herausspaltung des Ringes C als Succinimid-Derivat und dessen Identifikation als (2S,3S)-Enantiomer (vgl. Schema) eine auf den Ring-C-Bezirk beschrankte Teilaussage zu machen [l]. Die im Formelbild 1 bzw. 2 wiedergegebene absolute Konfiguration im

The reduction of methyl‐CoM to methane in methanogenic bacteria is dependent on a low‐M r, heat‐stable compound designated component B, the structure of which has recently been assigned as N‐7‐mercaptoheptanoyl‐O‐phosphothreonine. We report here that only the enantiomer derived from O‐phospho‐L‐threonine has cofactor activity. N‐7‐Mercaptoheptanoyl‐O‐phospho‐D‐threonine was neither active nor inhibitory.

Coenzyme F430 is a hydroporphinoid nickel complex present in all methanogenic bacteria. It is part of the enzyme system which catalyzes methane formation from methyl-coenzyme M. We describe here that under certain conditions a second nickel porphinoid accumulates in methanogenic bacteria. The compound was identified at 15,l 73-seco-F430-173-acid. The structural assignment rests on 14C-labelling experiments, fast-atom-bombardment mass spectra, 'H-NMR spectra of the corresponding hexamethyl ester, and ultraviolet/visible spectral comparison with model compounds. In cell extracts and in intact cells of methanogenic bactera, 15,173-seco-F430-173-acid was converted to F430. These findings indicate that the new nickel-containing porphinoid is an intermediate in the biosynthesis of coenzyme F430.Coenzyme F430 [l] is a yellow non-fluorescent nickel porphinoid found in all methanogenic bacteria [2 -51. Its structure has been recently determined [6-91. As shown in Fig. 1, coenzyme F430 1 possesses a highly saturated ligand system with a chromophore not previously encountered among natural tetrapyrroles. It may be considered a tetrahydro derivative of a corphin [6, 101, combining structural elements of both porphyrins and corrins. The isolated imine double bond, the lactam ring attached to ring B, and the sixmembered carbocyclic ring, built from the propionate side chain in ring D, further distinguish coenzyme F430 from other natural prophinoids. Suggestions that the parent coenzyme is larger, containing coenzyme M and a lumazine derivative covalently bound to the porphinoid ligand skeleton, in the meantime, have been ruled out [7, 11, 121. [19, 201. Coenzyme F430 is present in the cells also in a non-proteinbound, free form [7, 11, 211. Free F430 has been shown to be the precursor of bound F430 in the biosynthesis of methylcoenzyme M reductase [21]. The free and the enzyme-bound form (the latter determined after dissociation from the protein) have identical structures [7, 111. The biosynthesis of coenzyme F430 has been partially unravelled. The pathway starts from glutamate [22], which via glutamyl-tRNA and glutamate I-semialdehyde is converted to 5-aminolevulinic acid (6Ala) [23]. From there the biosynthesis proceeds along the well established pathway to uroporphyrinogen I11 [24], the common precursor of all natural tetrapyrroles [25]. Analogous ot the biosynthesis of vitamin BI2 and siroheme [25], uroporphyrinogen I11 is then methylated at positions 2 and 7 by S-adenosylmethionine [6, 261 in a sequence of reactions which, according to the available evidence, lead to 15,23-dihydro-sirohydrochlorin [9, 26 -281. The subsequent steps converting dihydro-sirohydrochlorin to coenzyme F430 and the intermediates involved are still unknown.In this communication we report the isolation of a nickelcontaining biosynthetic precursor of coenzyme F430 which was found to be a 15,173-seco derivative of F430, possessing a free propionate side chain in ring D in place of the sixmembered carbocyclic ring (3 Fig. 1). MATERIALS AND ...