Redox Properties of Flavocytochrome c₃ from Shewanella frigidimarina NCIMB400

Abstract

The thermodynamic and catalytic properties of flavocytochrome c3 from Shewanella frigidimarina have been studied using a combination of protein film voltammetry and solution methods. As measured by solution kinetics, maximum catalytic efficiencies for fumarate reduction (kcat/Km = 2.1 x 10(7) M-1 s-1 at pH 7.2) and succinate oxidation (kcat/Km = 933 M-1 s-1 at pH 8.5) confirm that flavocytochrome c3 is a unidirectional fumarate reductase. Very similar catalytic properties are observed for the enzyme adsorbed to monolayer coverage at a pyrolytic graphite "edge" electrode, thus confirming the validity of the electrochemical method for providing complementary information. In the absence of fumarate, the adsorbed enzyme displays a complex envelope of reversible redox signals which can be deconvoluted to yield the contributions from each active site. Importantly, the envelope is dominated by the two-electron signal due to FAD [E degrees ' = -152 mV vs the standard hydrogen electrode (SHE) at pH 7.0 and 24 degrees C] which enables quantitative examination of this center, the visible spectrum of which is otherwise masked by the intense absorption bands due to the hemes. The FAD behaves as a cooperative two-electron center with a pH-dependent reduction potential that is modulated (pKox at 6.5) by ionization of a nearby residue. In conjunction with the kinetic pKa values determined for the forward and reverse reactions (7.4 and 8.6, respectively), a mechanism for fumarate reduction, incorporating His365 and an anionic form of reduced FAD, is proposed. The reduction potentials of the four heme groups, estimated by analysis of the underlying envelope, are -102, -146, -196, and -238 mV versus the SHE at pH 7.0 and 24 degrees C and are comparable to those determined by redox potentiometry.

Identification of a Small Tetraheme Cytochrome c and a Flavocytochrome c as Two of the Principal Soluble Cytochromes c in Shewanella oneidensis Strain MR1

“…The redox potentials of the Shewanella fumarate reductase hemes, obtained by another method, average Ϫ170 mV and vary by as much as 136 mV (40). The higher redox potentials of the ST and FL cyt's are consistent with lower solvent exposure than in the cytochromes c 3 .…”

Purification and Magneto-optical Spectroscopic Characterization of Cytoplasmic Membrane and Outer Membrane Multiheme c-Type Cytochromes from Shewanella frigidimarina NCIMB400

“…Furthermore, several strains of S. putrefaciens have been demonstrated to contain menaquinones and methylmenaquinones (32), which would be expected to feature lower midpoint reduction potentials than ubiquinones. Regarding putative electron acceptors for CymA in S. frigidimarina NCIMB400, the heme midpoint potentials of the periplasmic tetraheme flavocytochrome c 3 fumarate reductase Fcc 3 have been reported as Ϫ102, Ϫ146, Ϫ196, and Ϫ238 mV (33). Our characterization has thus revealed CymA not to feature markedly lower potential hemes than the redox centers found in a protein it is proposed to reduce during a mode of anaerobic respiration.…”

Structure of adenylylsulfate reductase from the hyperthermophilic Archaeoglobus fulgidus at 1.6-Å resolution

“…6). Thus, the introduction of a seven-amino acid loop into the active site of an ancestral APS reductase could result in a planar isoalloxazine ring, concomitant with a negative shift in reduction potential as required for fumarate reduction (38). The comparison of APS and fumarate reductases provides an instructive example, by which means different biochemical reactions are accomplished by highly similar protein scaffolds mainly through the redesign of loop structures.…”