Perspective | Published:

The plasticity of redox cofactors: from metalloenzymes to redox-active DNA

Nature Reviews Chemistryvolume 2pages231243 (2018) | Download Citation


Metal cofactors considerably widen the catalytic space of naturally occurring enzymes whose specific and enantioselective catalytic activity constitutes a blueprint for economically relevant chemical syntheses. To optimize natural enzymes and uncover novel reactivity, we need a detailed understanding of cofactor–protein interactions, which can be challenging to obtain in the case of enzymes with sophisticated cofactors. As a case study, we summarize recent research on the [FeFe]-hydrogenases, which interconvert protons, electrons and dihydrogen at a unique iron-based active site. We can now chemically synthesize the complex cofactor and incorporate it into an apo-protein to afford functional enzymes. By varying both the cofactor and the polypeptide components, we have obtained detailed knowledge on what is required for a metal cluster to process H2. In parallel, the design of artificial proteins and catalytically active nucleic acids are advancing rapidly. In this Perspective, we introduce these fields and outline how chemists and biologists can use this knowledge to develop novel tailored semisynthetic catalysts.

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The authors appreciate funding from the VolkswagenStiftung (Design of [FeS]-cluster containing Metallo-DNAzymes (Az 93412)) and the Deutsche Forschungsgemeinschaft (DFG) (the Cluster of Excellence RESOLV EXC1069 and GRK 2341: Microbial Substrate Conversion).

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  1. Faculty of Biology and Biotechnology, Department of Plant Biochemistry, Workgroup Photobiotechnology, Ruhr-University of Bochum, Bochum, Germany

    • Anja Hemschemeier
    •  & Thomas Happe


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All authors contributed to researching the article, discussing the content and writing and editing of the article.

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The authors declare no competing interests.

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Correspondence to Anja Hemschemeier or Thomas Happe.



An enzyme that hydrolyses β-lactam-containing antibiotics, such as penicillins or cephalosporins. Most β-lactam antibiotics inhibit biosynthesis of the bacterial cell wall. Bacteria expressing genes that encode for β-lactamase are resistant to such antibiotics.

Chlorophycean microalgae

The Chlorophyta (green algae) diverged from land plants over a billion years ago. Some unicellular green algae, such as the model organism Chlamydomonas reinhardtii, synthesize [FeFe]-hydrogenase enzymes that are located within their chloroplasts — the organelles in which photosynthesis takes place. In C. reinhardtii, [FeFe]-hydrogenases accept electrons from ferredoxin, which, in turn, can be reduced by photosynthetic electron transport. The alga can thus dispose of excess light energy through H+ reduction.

Directed evolution

A molecular biology technique that mimics the process of natural evolution — mutagenesis and selection. A sequence that encodes a protein whose characteristics are to be altered is randomly mutagenized (for example, by error-prone PCR), and the resulting proteins are screened for optimized features, such as optimum temperature or solvent stability. Several rounds of mutagenesis and selection are usually performed.

Entatic state

Translated literally, entatic means under tension or stretched. In the case of metalloenzymes, the ‘entatic state’ refers to an unusual geometric or electronic configuration of a metal ion that would not occur in the presence of unrestrained ligands. This poised state is enforced by the protein and enables the protein-specific reactivity of a given metal centre.

Error-prone PCR

The DNA sequence that codes for a protein can be randomly mutated by modulating the PCR conditions such that DNA polymerase introduces mistakes into the newly polymerized DNA strand. Thereby, random changes in the amino acid composition of the encoded protein are generated. The technique is usually coupled with (high-throughput) screening techniques to identify proteins with desired variations in their properties.

Genetic code expansion

One can enable organisms to introduce unnatural amino acid residues into proteins using their natural translation machinery. For this purpose, a novel (orthogonal) tRNA is evolved together with its aminoacyl-tRNA synthetase, which couples the respective (unnatural) amino acid to the tRNA. The tRNA is designed to recognize a stop codon, which can then be introduced into the sequence that encodes the protein whose amino acid content is to be changed. Most often, the rare amber stop codon is utilized.


A specific structure of guanine-rich nucleic acids in which so-called G-tetrads are stacked. G-tetrads are built from four guanine bases that form a square planar structure.


These enzymes can oxidize H2 or produce H2 from protons. Organisms utilize hydrogenases to make use of the energy content of H2 (for example, through the Knallgas reaction) or to dispose of excess electrons (for example, during fermentation).

Pleiotropic effects

This term was originally used to describe genes that had more than one effect. However, its general meaning in biology refers to something that has multiple, often unrelated impacts. The exchange of an amino acid in a protein by site-directed mutagenesis can have a structural effect, for example, when the two residues differ in size, or a functional effect, when the chemical properties of the residues differ. If both effects are observed, the mutation is said to have pleiotropic effects.

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