Matthew J. Ryle

Non-heme iron oxygenases.

Our understanding of the biological significance and chemical properties of non-heme iron oxygenases has increased dramatically in recent years. New group members have emerged from genome sequences and biochemical analyses. Spectroscopic and crystallographic studies have provided critical insights into catalysis. Self-hydroxylation reactions, commonplace in these proteins, reveal important features of metallocenter reactivity.

Interconversion of two oxidized forms of taurine/α-ketoglutarate dioxygenase, a non-heme iron hydroxylase: Evidence for bicarbonate binding

Taurine/α-ketoglutarate (αKG) dioxygenase, or TauD, is a mononuclear non-heme iron hydroxylase that couples the oxidative decarboxylation of αKG to the decomposition of taurine, forming sulfite and aminoacetaldehyde. Prior studies revealed that taurine-free TauD catalyzes an O2- and αKG-dependent self-hydroxylation reaction involving Tyr-73, yielding an Fe(III)-catecholate chromophore with a λmax of 550 nm. Here, a chromophore (λmax 720 nm) is described and shown to arise from O2-dependent self-hydroxylation of TauD in the absence of αKG, but requiring the product succinate. A similar chromophore rapidly develops with the alternative oxidant H2O2. Resonance Raman spectra indicate that the ≈700-nm chromophore also arises from an Fe(III)-catecholate species, and site-directed mutagenesis studies again demonstrate Tyr-73 involvement. The ≈700-nm and 550-nm species are shown to interconvert by the addition or removal of bicarbonate, consistent with the αKG-derived CO2 remaining tightly bound to the oxidized metal site as bicarbonate. The relevance of the metal-bound bicarbonate in TauD to reactions of other members of this enzyme family is discussed.

Self-hydroxylation of taurine/α-ketoglutarate dioxygenase: evidence for more than one oxygen activation mechanism

Abstract2-Aminoethanesulfonic acid (taurine)/α-ketoglutarate (αKG) dioxygenase (TauD) is a mononuclear non-heme iron enzyme that catalyzes the hydroxylation of taurine to generate sulfite and aminoacetaldehyde in the presence of O2, αKG, and Fe(II). Fe(II)TauD complexed with αKG or succinate, the decarboxylated product of αKG, reacts with O2 in the absence of prime substrate to generate 550- and 720-nm chromophores, respectively, that are interconvertible by the addition or removal of bound bicarbonate and have resonance Raman features characteristic of an Fe(III)–catecholate complex. Mutagenesis studies suggest that both reactions result in the self-hydroxylation of the active-site residue Tyr73, and liquid chromatography nano-spray mass spectrometry/mass spectrometry evidence corroborates this result for the succinate reaction. Furthermore, isotope-labeling resonance Raman studies demonstrate that the oxygen atom incorporated into the tyrosyl residue derives from H218O and 18O2 for the αKG and succinate reactions, respectively, suggesting distinct mechanistic pathways. Whereas the αKG-dependent hydroxylation likely proceeds via an Fe(IV)=O intermediate that is known to be generated during substrate hydroxylation, we propose Fe(III)–OOH (or Fe(V)=O) as the oxygenating species in the succinate-dependent reaction. These results demonstrate the two oxygenating mechanisms available to enzymes with a 2-His-1-carboxylate triad, depending on whether the electron source donates one or two electrons.

ADP-Ribosylation of Variants of Azotobacter vinelandii Dinitrogenase Reductase by Rhodospirillum rubrum Dinitrogenase Reductase ADP-Ribosyltransferase

In a number of nitrogen-fixing bacteria, nitrogenase is posttranslationally regulated by reversible ADP-ribosylation of dinitrogenase reductase. The structure of the dinitrogenase reductase from Azotobacter vinelandii is known. In this study, mutant forms of dinitrogenase reductase from A. vinelandii that are affected in various protein activities were tested for their ability to be ADP-ribosylated or to form a complex with dinitrogenase reductase ADP-ribosyltransferase (DRAT) from Rhodospirillum rubrum. R140Q dinitrogenase reductase could not be ADP-ribosylated by DRAT, although it still formed a cross-linkable complex with DRAT. Thus, the Arg 140 residue of dinitrogenase reductase plays a critical role in the ADP-ribosylation reaction. Conformational changes in dinitrogenase reductase induced by an F135Y substitution or by removal of the Fe(4)S(4) cluster resulted in dinitrogenase reductase not being a substrate for ADP-ribosylation. Through cross-linking studies it was also shown that these changes decreased the ability of dinitrogenase reductase to form a cross-linkable complex with DRAT. Substitution of D129E or deletion of Leu 127, which result in altered nucleotide binding regions of these dinitrogenase reductases, did not significantly change the interaction between dinitrogenase reductase and DRAT. Previous results showed that changing Lys 143 to Gln decreased the binding between dinitrogenase reductase and dinitrogenase (L. C. Seefeldt, Protein Sci. 3:2073-2081, 1994); however, this change did not have a substantial effect on the interaction between dinitrogenase reductase and DRAT.