Abstract
The class of [NiFe]–hydrogenases is characterized by a bimetallic cofactor comprising low–spin nickel and iron ions, the latter of which is modified with a single carbon monoxide (CO) and two cyanide (CN−) molecules. Generation of these ligands in vivo requires a complex maturation apparatus in which the HypC–HypD complex acts as a ‘construction site’ for the Fe–(CN)2CO portion of the cofactor. The order of addition of the CO and CN– ligands determines the ultimate structure and catalytic efficiency of the cofactor; however much debate surrounds the succession of events. Here, we present an FT–IR spectroscopic analysis of HypC–HypD isolated from a hydrogenase–competent wild–type strain of Escherichia coli. In contrast to previously reported samples, HypC–HypD showed spectral contributions indicative of an electron–rich Fe–CO cofactor, at the same time lacking any Fe–CN– signatures. This immature iron site binds external CO and undergoes oxidative damage when in contact with O2. Binding of CO protects the site against loss of spectral features associated with O2 damage. Our findings strongly suggest that CO ligation precedes cyanation in vivo. Furthermore, the results provide a rationale for the deleterious effects of O2 on in vivo cofactor biosynthesis.
Highlights
Hydrogenases are ancient and widespread iron–sulfur enzymes [1]
HypCDBL exhibits contributions from two bands at 2072 and 2096 cm21 and a single contribution at 1954 cm21. The latter signal has been attributed to Fe(II)–carbon monoxide (CO), while both former bands stem from Fe(II)–CN1 and Fe(II)–CN2 [17,18]
The novel population isolated from the HypCDMC mixed–state sample reacts with O2 and binds external CO
Summary
Hydrogenases are ancient and widespread iron–sulfur enzymes [1]. All hydrogenases exploit low–spin transition metal ions to catalyze proton reduction and heterolytic dihydrogen oxidation following H2 « H+ + H2 « 2 H+ + 2 e2. A common motif in the organometallic redox chemistry of [NiFe]– and [FeFe]– hydrogenases is a modification of the active site cofactor with carbon monoxide (CO) and cyanide (CN2). These are found coordinated exclusively with the iron ions and allow for efficient hydrogen turnover at minimal overpotential [4]. The catalytic cofactor of [NiFe]–hydrogenases consists of a nickel ion and an iron ion. The sixth coordination site in the iron ion is shared with nickel and was found to be occupied by hydrogen and oxygen species [3,6,7]. We refer to the Ni–Fe cofactor as ‘[NiFe]–(CN)2CO’ in the following
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