Conserved Histidine Adjacent to the Proximal Cluster Tunes the Anaerobic Reductive Activation of Escherichia coli Membrane-Bound [NiFe] Hydrogenase-1

Abstract

[NiFe] hydrogenases are electrocatalysts that oxidize H₂ at a rapid rate without the need for precious metals. All membrane-bound [NiFe] hydrogenases (MBH) possess a histidine residue that points to the electron-transfer iron sulfur cluster closest ("proximal") to the [NiFe] H₂-binding active site. Replacement of this amino acid with alanine induces O₂ sensitivity, and this has been attributed to the role of the histidine in enabling the reversible O₂-induced over-oxidation of the [Fe₄S₃Cys₂] proximal cluster possessed by all O₂-tolerant MBH. We have created an Escherichia coli Hyd-1 His-to-Ala variant and report O₂-free electrochemical measurements at high potential that indicate the histidine-mediated [Fe₄S₃Cys₂] cluster-opening/closing mechanism also underpins anaerobic reactivation. We validate these experiments by comparing them to the impact of an analogous His-to-Ala replacement in Escherichia coli Hyd-2, a [NiFe]-MBH that contains a [Fe₄S₄] center.

Keywords: bioinorganic chemistry; electrochemistry; electron transfer; hydrogen; metalloenzymes.

Figure 1

Structural similarities and differences between O₂‐tolerant MBH and O₂‐sensitive [NiFe] hydrogenases. i) Cartoon of the heterodimeric minimal functional unit. ii) The difference between the proximal iron sulfur cluster (FeS_proximal) structure of O₂‐sensitive enzymes which contain an [Fe₄S₄] center, and the proximal [Fe₄S₃Cys₂] center of O₂‐tolerant MBH, which can reversibly interconvert between a “closed”, +3, reduced state and an “open”, +5, “over‐oxidized” state. The relative position of His amino acids to these centers is highlighted, with E. coli Hyd‐2 numbering used in the O₂‐sensitive structure which is derived from PDB 3MYR^[7], and E. coli Hyd‐1 numbering used in the O₂‐tolerant structures which are derived from PDB 3RGW4 for the +5 state and PDB 4IUC5 for the +3 state. Colors: gray ribbon, small subunit; green ribbon, large subunit; yellow spheres and sticks, sulfur; orange spheres, iron; light blue, carbon; dark blue, nitrogen; red, oxygen. iii) Dance's proposed6 proton‐transfer pathway from Glu‐73 to S3 that is mediated by His‐229, which is sensitive to the active site geometry via its H‐bond to Thr‐80. Top structure based on E. coli Hyd‐1 PDB3UQY, and bottom on E. coli Hyd‐1 3USC. Green labels indicate large subunit (HyaB) residues, gray labels indicate small subunit (HyaA) residues and the location of the OH⁻ group observed by Frielingsdorf et al.5 is indicated. Green spheres are used to indicate nickel, all other color Scheme details are as in (ii). iv) Sequence alignments comparing the ligation of the proximal cluster of O₂‐tolerant MBH and O₂‐sensitive [NiFe] hydrogenase. E. coli Hyd‐1 numbering is used.

Scheme 1

How the catalytically active Ni²⁺ form of the active site, the Ni‐Si_a state, can be reversibly inactivated to form the Ni³⁺‐containing Ni‐B state.

Figure 2

Catalytic voltammograms (5 mV s⁻¹) measured under an atmosphere of 3 % H₂, 97 % N₂ for native Hyd‐1 (top left), Hyd1‐H229A variant (bottom left), native Hyd‐2 (top right), and Hyd2‐H214A variant (bottom right). Within each panel, the same film of enzyme was used for all the pH points. Four scans were measured at each pH and the fourth cycle is shown, with the current traces normalized to the maximum H₂ oxidation current.

Figure 3

Chronoamperometric traces showing the O₂ tolerance of native Hyd‐2 (black) and the Hyd2‐H214A variant (red) at pH 6.0 (top), as well as native Hyd‐1 (bottom left) and the Hyd1‐H229A variant (bottom right) at pH 4.5, 6.0 and 7.6, as indicated. For the Hyd‐2 experiments, the H₂ oxidation activity was monitored at a constant potential of −0.135 V vs SHE; for the Hyd‐1 experiments, the potentials were +0.175 V vs SHE (pH 4.5), +0.085 V vs SHE (pH 6.0) or −0.05 V vs SHE (pH 7.6). Current traces were corrected for film loss and normalized to the H₂ oxidation current immediately before the addition of O₂.

Figure 4

Potential step experiment to measure activation and inactivation rates of native Hyd‐1 and variant H1‐H229A at pH 6.0, 10 % H₂. The potential‐time steps applied in the chronoamperometry experiment (top). Resulting chronoamperometric traces for native Hyd‐1 (middle) and variant Hyd1‐H229A (bottom). Other experimental conditions: 37 °C, electrode rotation rate 4,000 rpm, N₂ carrier gas and total gas flow rate 1,000 scc min⁻¹.

Figure 5

Inactivation, k _I, and activation, k _A, rate constant values for native Hyd‐1 and variant Hyd1‐H229A, as extracted from analysis of pH 6.0, 10 % H₂ experiments such as those shown in Figure S6. Data points show the average value calculated from three repeat experiments, and error bars show the standard error.