Issue 21, 2020

The large subunit of the regulatory [NiFe]-hydrogenase from Ralstonia eutropha – a minimal hydrogenase?

Abstract

Chemically synthesized compounds that are capable of facilitating the reversible splitting of dihydrogen into protons and electrons are rare in chemists' portfolio. The corresponding biocatalysts – hydrogenases – are, however, abundant in the microbial world. [NiFe]-hydrogenases represent a major subclass and display a bipartite architecture, composed of a large subunit, hosting the catalytic NiFe(CO)(CN)2 cofactor, and a small subunit whose iron–sulfur clusters are responsible for electron transfer. To analyze in detail the catalytic competence of the large subunit without its smaller counterpart, we purified the large subunit HoxC of the regulatory [NiFe]-hydrogenase of the model H2 oxidizer Ralstonia eutropha to homogeneity. Metal determination and infrared spectroscopy revealed a stoichiometric loading of the metal cofactor. This enabled for the first time the determination of the UV-visible extinction coefficient of the NiFe(CO)(CN)2 cofactor. Moreover, the absence of disturbing iron–sulfur clusters allowed an unbiased look into the low-spin Fe2+ of the active site by Mössbauer spectroscopy. Isolated HoxC was active in catalytic hydrogen–deuterium exchange, demonstrating its capacity to activate H2. Its catalytic activity was drastically lower than that of the bipartite holoenzyme. This was consistent with infrared and electron paramagnetic resonance spectroscopic observations, suggesting that the bridging position between the active site nickel and iron ions is predominantly occupied by water-derived ligands, even under reducing conditions. In fact, the presence of water-derived ligands bound to low-spin Ni2+ was reflected by the absorption bands occurring in the corresponding UV-vis spectra, as revealed by time-dependent density functional theory calculations conducted on appropriate in silico models. Thus, the isolated large subunits indeed represent simple [NiFe]-hydrogenase models, which could serve as blueprints for chemically synthesized mimics. Furthermore, our data point to a fundamental role of the small subunit in preventing water access to the catalytic center, which significantly increases the H2 splitting capacity of the enzyme.

Graphical abstract: The large subunit of the regulatory [NiFe]-hydrogenase from Ralstonia eutropha – a minimal hydrogenase?

Supplementary files

Article information

Article type
Edge Article
Submitted
06 Mar 2020
Accepted
24 Apr 2020
First published
27 Apr 2020
This article is Open Access

All publication charges for this article have been paid for by the Royal Society of Chemistry
Creative Commons BY-NC license

Chem. Sci., 2020,11, 5453-5465

The large subunit of the regulatory [NiFe]-hydrogenase from Ralstonia eutropha – a minimal hydrogenase?

G. Caserta, C. Lorent, A. Ciaccafava, M. Keck, R. Breglia, C. Greco, C. Limberg, P. Hildebrandt, S. P. Cramer, I. Zebger and O. Lenz, Chem. Sci., 2020, 11, 5453 DOI: 10.1039/D0SC01369B

This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. You can use material from this article in other publications, without requesting further permission from the RSC, provided that the correct acknowledgement is given and it is not used for commercial purposes.

To request permission to reproduce material from this article in a commercial publication, please go to the Copyright Clearance Center request page.

If you are an author contributing to an RSC publication, you do not need to request permission provided correct acknowledgement is given.

If you are the author of this article, you do not need to request permission to reproduce figures and diagrams provided correct acknowledgement is given. If you want to reproduce the whole article in a third-party commercial publication (excluding your thesis/dissertation for which permission is not required) please go to the Copyright Clearance Center request page.

Read more about how to correctly acknowledge RSC content.

Social activity

Spotlight

Advertisements