Direct detection and measurement of electron relays in a multicentered enzyme: Voltammetry of electrode-surface films of E-coli fumarate reductase, an iron-sulfur flavoprotein

Intramolecular electron relays operating in a multicentered enzyme are revealed by protein film voltammetry. The membrane-extrinsic catalytic domain of E. coli fumarate reductase (FrdAB) adsorbs to electroactive monolayer coverage at a rotating pyrolytic graphite edge electrode, giving characteristic voltammetric signals that are resolved and assigned to redox-active sites. At pH 7.3 (2 degrees C) signals attributable to Centers 1 ([2Fe-2S]) and 3 ([3Fe-4S]) and FAD are enveloped together around -50 mV, while Center 2 ([4Fe-4S]) appears as a weaker signal at -305 mV. At pH 9.5, similar voltammetry is observed, the main difference being that the FAD component shifts to the negative edge of the enveloper. The prominence of the two-electron FAD signal enables active-site redox transformations to be tracked and examined over a range of conditions. Scans at rates up to 20 V s(-1) in the absence of fumarate shaw that electrons are relayed to the FAD, most obviously by Centers 1 and 3. Upon adding fumarate, the signals undergo transformations ss specific centers engage in catalytic electron transport. A sigmoidal wave originating in the FAD envelope region is joined by a second wave close to the potential of Center 2. This is particularly evident under conditions optimizing enzyme catalytic control (as opposed to mass-transport control), i.e. high fumarate levels, high rotation rate, and pH 9.0 at which the enzyme is less active than at pH 7.0. Intramolecular electron transport is partitioned between different relay systems depending on catalytic demand and proficiency of the FAD as electron acceptor. At high pH, the less favorable driving force for electron transfer from Centers 1 and 3 places a greater burden on Center 2. Catalytic voltammograms show hysteresis in the presence of oxalacetate, and inhibitor binding preferentially to oxidized FAD. Reductive activation is slow but accelerates sharply below the potential of Center 2, showing that this cluster is much more effective than the others in reducing the inhibitor-bound active site. The results demonstrate how voltammetry can be used to quantify intramolecular electron transfer among multiple sites in complex enzymes.