Reconsidering the Enzyme Kinetics of [FeFe]-Hydrogenases: Improved Turnover Rates and New Insights into pH and Potential Dependence with Eu(II)-Based Solution Assays

TL;DR

This paper introduces a new method using Eu(II) to study hydrogenase enzyme kinetics, revealing higher activity and clearer pH dependence than traditional methods.

Contribution

A novel Eu(II)-based assay method is introduced, offering improved turnover rates and resolving pH-related discrepancies in hydrogenase studies.

Findings

Eu(II) provides a larger potential window and higher turnover frequencies than sodium dithionite.

Hydrogenase activity peaks at pH 5–6 with Eu(II), aligning better with electrochemical data.

A 180 mV potential change increases catalytic rate by 35-fold, surpassing prior reports.

Abstract

Metal-dependent redox enzymes are central for microbial
processing
of gases, as exemplified by hydrogenase, nitrogenase, and carbon monoxide
dehydrogenase. Due to their remarkable efficiencies and high biotechnological
relevance, such gas-processing enzymes are intensively studied. Nevertheless,
many of their mechanistic details remain opaque. We herein report
a new method for solution assays under reducing conditions based on
europium­(II) as a terminal reductant and show how it can be employed
to gain new insight into hydrogenase kinetics. Compared with the commonly
used reductant sodium dithionite, this work shows that Eu­(II) can
serve as a robust and relatively easy-to-handle alternative electron
donor, also providing a larger potential window for catalytic studies.
Further, this work clarifies previous discrepancies in the literature
regarding the influence of pH on hydrogenase kinetics in these assays.
Our study shows that sodium dithionite, most likely due to its decomposition
into SO2, alters hydrogenase kinetics in solution assays.
Using [FeFe]-hydrogenase I from Clostridium pasteurianum (CpI) as a model system, Eu­(II)-based solution assays demonstrated
a pH optimum of 5–6 and rates greatly exceeding those observed
with sodium dithionite assays. The higher turnover frequencies observed
at low pH obtained with Eu­(II) align more closely with the electrochemical
data. Additionally, a strong driving force dependency was identified.
A solution potential change of approximately 180 mV resulted in a
35-fold increase in the catalytic rate, yielding activities far surpassing
those of earlier reports on CpI turnover frequencies. These findings
provide new insight into the pH dependence and overall kinetic performance
of [FeFe]-hydrogenases. More broadly, the report outlines alternative
assay methods employing Eu­(II) to better understand the enzyme kinetics
of hydrogenases and related metalloenzymes.

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