A Radical Intermediate in Tyrosine Scission to the CO and CN ⁻ Ligands of FeFe Hydrogenase

Abstract

The radical S-adenosylmethionine (SAM) enzyme HydG lyses free l-tyrosine to produce CO and CN(-) for the assembly of the catalytic H cluster of FeFe hydrogenase. We used electron paramagnetic resonance spectroscopy to detect and characterize HydG reaction intermediates generated with a set of (2)H, (13)C, and (15)N nuclear spin-labeled tyrosine substrates. We propose a detailed reaction mechanism in which the radical SAM reaction, initiated at an N-terminal 4Fe-4S cluster, generates a tyrosine radical bound to a C-terminal 4Fe-4S cluster. Heterolytic cleavage of this tyrosine radical at the Cα-Cβ bond forms a transient 4-oxidobenzyl (4OB(•)) radical and a dehydroglycine bound to the C-terminal 4Fe-4S cluster. Electron and proton transfer to this 4OB(•) radical forms p-cresol, with the conversion of this dehydroglycine ligand to Fe-bound CO and CN(-), a key intermediate in the assembly of the 2Fe subunit of the H cluster.

CO and CN − syntheses by [FeFe]-hydrogenase maturase HydG are catalytically differentiated events

“…2. Its Cα-Cβ bond is cleaved after H-abstraction at the amino group by the SAM-derived 5′-deoxyadenosine radical, generating a p-cresyl radical (24) and dehydroglycine (DHG). 3.…”

Metallocofactor assembly for [FeFe]-hydrogenases

“…The last two are incorporated into the [2Fe] H subcluster [15] together with HydG-derived iron atoms [16] The intermediate dehydroglycine is converted to CO and cyanide, a reaction that likely involves a second FeS cluster present in HydG, termed the auxiliary cluster [17]. The binding of these ligands to the auxiliary cluster was first reported using time resolved FTIR spectroscopy, which identified two further intermediates (Figure 1 c), complex A, formed after ~30 seconds, which is subsequently converted to complex B [12]. The presence of a second CO ligand in complex B implies a likely requirement for the fragmentation of two dehydroglycine equivalents to form this intermediate.…”

“…The Nterminal cluster consistently showed EPR properties typical of the cluster used for radical SAM chemistry, [4Fe-4S] RS [12,17,18], however more variation in the spectroscopic properties for the Cterminal auxiliary cluster have been reported. It seems likely that differences in the enzyme source, purification method and whether the clusters were subject to chemical reconstitution (addition of exogenous Fe(II) and sulfide after purification) contributed to these spectroscopic discrepancies.…”

“…Structural studies with a related radical SAM enzyme [11] suggest that in HydG the hydrogen atom is abstracted from the α-nitrogen of tyrosine, followed by β-scission of the tyrosine Cα-Cβ bond (Figure 1c). The resultant hydroxybenzyl radical has been characterized in detail using tyrosine isotopologues and freeze quench EPR spectroscopy [12]. Biochemical experiments with HydG identified three tyrosine derived products: the byproduct p-cresol [7], cyanide [13] and carbon monoxide [14].…”

“…It seems likely that differences in the enzyme source, purification method and whether the clusters were subject to chemical reconstitution (addition of exogenous Fe(II) and sulfide after purification) contributed to these spectroscopic discrepancies. For example, the auxiliary clusters of HydGs from Clostridium acetobutylicum (Ca) [17] and Thermotoga maritima (Tm) [18] have spectroscopic properties of a [4Fe-4S] + cluster, whereas EPR analysis of the enzyme from Shewanella oneidensis (So) upon isolation contained predominantly a high spin FeS auxiliary cluster that upon turnover in the presence of tyrosine, SAM and dithionite converted to a [4Fe-4S] + form [12].…”