Metallocofactors that Activate Small Molecules

Nitrogenase, the primary biological source of fixed nitrogen, has been studied by various biochemical and biophysical methods to determine the mechanism of nitrogen reduction to ammonia. Previously, structural studies have contributed to determining the arrangement and identity of the unique metallocofactors of the as-isolated nitrogenase enzyme. Due to the multi-protein, dynamic nature of catalysis in nitrogenase, structurally capturing intermediates is not trivial. Recently, we have developed methods for preparing crystallographic samples of nitrogenase from active assay mixtures. The “out-of-assay” approach has yielded structures of small molecules bound to the active site cofactor, revealing an unexpected rearrangement of the belt sulfur atoms. The activity-based methods provide a framework for accessing non-resting states of the cofactor and introduce new questions surrounding the controlled binding and release of substrates. In the following, we discuss recent structural advances in the field and the novel directions for future activity-based research.

Nitrogenase catalyzes the remarkable chemical transformations of N₂ to NH₃, and C₁ substrates to hydrocarbons, under ambient conditions. The best-studied Mo-nitrogenase utilizes a complex metallocofactor ([MoFe₇S₉C(R-homocitrate)]) for substrate binding and reduction; however, the complexity of this cofactor has hindered a better understanding of its mechanistic details and chemical synthesis so far. Driven by the pressing questions related to the structure and function of the nitrogenase cofactor, research in the past decades has been focused on unraveling the biosynthetic mechanism of this metallocluster in order to cultivate knowledge of how the cofactor is functionalized in this process. In this review, we summarize the recent advances toward a better understanding of the biosynthesis of the nitrogenase cofactor, with a particular focus on the biosynthetic events related to the generation of its unique core structure. Information derived from these studies has unveiled a novel, radical SAM-dependent mechanism of carbide insertion that orchestrates the coupling and rearrangement of two 4Fe cluster modules into a unique 8Fe cofactor core, as well as a sulfite-based route that incorporates a ‘9th sulfur’ at the catalytically important “belt” region of the cofactor. Continued efforts along this line of investigation will further unravel the biosynthetic mechanism of the nitrogenase cofactor while facilitating investigations into the elusive catalytic mechanism of nitrogenase.

The only enzyme that is able to fix nitrogen, nitrogenase, reduces inert and abundant dinitrogen (N₂) into bioavailable ammonia (NH₃) under ambient conditions. The most investigated variant, the MoFe nitrogenase, uses three metallo-cofactors: the [Fe₄S₄] cluster in the electron-carrier component (Fe protein), as well as the [Fe₈S₇] (P-cluster) and [MoFe₇S₉C] (M-cluster) clusters in the catalytic component (MoFe protein). To better understand the physical properties of these cofactors, various methods have been developed for the chemical synthesis of model metal-sulfur clusters. In this review, we address the following topics with emphasis on recent developments: (a) the synthesis of all-ferrous [Fe₄S₄]⁰ clusters, which are isoelectronic to the super-reduced state of the cluster in the Fe protein, (b) the reproduction of the unique [Fe₈S₇] inorganic core of the P-cluster, and (c) the synthesis of metal-sulfur clusters relevant to the M-cluster and their variants that incorporate a light atom. Even though reproduction of the M-cluster remains elusive, some recent advances seem promising toward new classes of metal-sulfur clusters that satisfy the key structural features of the M-cluster.

Molybdenum and tungsten are, respectively, the only second and third transition metal ions with well-defined functions in living organisms and with a single exception are found in association with a novel pyranopterin dithiolene cofactor called molybdopterin. This review focusses on the catalytic mechanisms of the molybdenum and tungsten enzymes, with an emphasis on the molybdenum and tungsten sites. Most, but not all, of the enzymes catalyze oxygen atom transferase redox chemistry, with the metal cycling between M(VI) and M(IV) formal oxidation states during the catalytic cycle. We discuss the range of reactions and what is known of mechanism for both oxo-transferase and non-oxo-transferase molybdenum and tungsten enzymes.

An overview of the pyranopterin dithiolene (MPT) component of the molybdenum cofactor (Moco) and how MPT may contribute to enzymatic catalysis is presented. The chapter begins with a brief review of MPT and Moco biosynthesis and continues to explore the nature of what is arguably the most electronically complex ligand in biology. To explore this complexity, we have dissected MPT into its relevant molecular components. These include the redox-active ene-1,2-dithiolate (dithiolene) and pterin moieties, which are bridged by a pyran that may be found in ring-opened or ring-closed configurations. The various redox possibilities of MPT bound to Mo are presented, along with the electronic structure of the redox components. MPTs are found to display a remarkable conformational variance in pyranopterin Mo enzymes. This is discussed in terms of a relationship to enzyme function and the potential for the observed non-planer distortions to reflect different MPT oxidation and tautomeric states. The chapter ends with a series of case studies featuring model compounds that highlight how biomimetic small molecule studies have contributed to furthering our understanding of the roles this remarkable ligand plays in the catalytic cycles of the enzymes.

Carbon monoxide dehydrogenases catalyze the reversible oxidation of CO with water to CO₂, two protons and two electrons. Phylogenetically diverse bacteria and archaea living under anaerobic conditions employ different classes of Ni,Fe-containing carbon monoxide dehydrogenases to use CO as an energy source or to contribute in converting CO₂ to acetyl-CoA.

The active site of carbon monoxide dehydrogenases contains a unique [NiFe₄S₄]-cluster, the only known example in nature where Ni is integrated into a heterocubane structure. The Ni ion serves as the catalytic nucleophilic center for activating CO and CO₂, in which it is supported by an electrophilic Fe ion placed in exo to the heterocubane cluster.

This review gives an overview on current ideas how Ni,Fe-containing carbon monoxide dehydrogenases reversibly oxidize CO to CO₂, with a focus on recent structural studies of the enzymes.