Search

Ferredoxin (Fd) is a small metalloprotein holding one or two Fe-S clusters in its inner shell. Like many other metalloproteins, Fd is redox active and involved in electron transfer during cellular metabolism. The electrons from reduced Fd are mostly used to regenerate NADPH under physiological conditions. Increasing number of attempts have been reported, however, where Fd delivers electrons to enable biosynthesis of valuable compounds. Various compounds ranging from H2 to vitamin D3 have been synthesized successfully using electrons mediated by Fd molecules. In this review, we provide an overview of the engineering studies utilizing Fd for biosynthesis of targeted molecules. The emphasis is on the role and activity of Fd as well as the methods used to improve the rate of electron transfer. Both microbial and electrochemical biosynthesis technologies are described and compared with respect to productivity and the compound being produced. In addition to the ferredoxins from the microbial organisms, artificially designed de novo types are described, highlighting the potential of the emerging computational methods used in metabolic and protein engineering. We believe that the recent advances in utilization of Fd for biosynthesis can result in breakthrough innovation across the biotechnology industry. [ABSTRACT FROM AUTHOR]

Biological H 2 production is one of the alternative technologies for producing H 2 in a renewable and sustainable fashion. The technology often relies on the [FeFe] hydrogenase enzyme for catalyzing the H 2 production reaction from protons and electrons as it is highly efficient. The high O 2 sensitivity of the enzyme have been one of the key obstacles for implementing the technology, but recent findings showed the feasibility of improving O 2 tolerance via protein engineering. In this study, we investigated the changes in the O 2 tolerance and aerobic H 2 production activity of an [FeFe] hydrogenase by replacing methionines on the surface with leucines. None of the mutations were detrimental to protein folding, and a few were able to exert either positive or negative influence on the O 2 tolerance of the enzyme. We combined the mutation exhibiting the highest improvement in O 2 tolerance with the rest of the Met – Leu replacements; however, the results indicated non-synergistic impact on the tolerance. Combinations with the other types of mutations that have been reported previously also failed to further improve the O 2 tolerance of the hydrogenase. These results provide new insights on how mutagenesis affects the enzyme's functional properties, as well as the molecular mechanism(s) behind the tolerance. • A novel mutation that enhances the O 2 tolerance of the [FeFe] hydrogenase. • Analysis of the combinatorial mutagenesis' influence on the key functional properties. • Protein engineering work employing both in silico and experimental methods. [ABSTRACT FROM AUTHOR]

Hydrogenases are metalloproteins capable of catalyzing the interconversion between molecular hydrogen and protons and electrons. The iron–sulfur clusters within the enzyme enable rapid relay of electrons which are either consumed or generated at the active site. Their unparalleled catalytic efficiency has attracted attention, especially for potential use in H2 production and/or fuel cell technologies. However, there are limitations to using hydrogenases, especially due to their high O2 sensitivity. The subclass, called [FeFe] hydrogenases, are particularly more vulnerable to O2 but proficient in H2 production. In this review, we provide an overview of mechanistic and protein engineering studies focused on understanding and enhancing O2 tolerance of the enzyme. The emphasis is on ongoing studies that attempt to overcome O2 sensitivity of the enzyme while it catalyzes H2 production in an aerobic environment. We also discuss pioneering attempts to utilize the enzyme in biological H2 production and other industrial processes, as well as our own perspective on future applications. [ABSTRACT FROM AUTHOR]