MacA is a Second Cytochrome c Peroxidase of Geobacter sulfurreducens

Abstract

The metal-reducing δ-proteobacterium Geobacter sulf urreducens produces a large number of c-type cytochromes, many of which have been implicated in the transfer of electrons to insoluble metal oxides. Among these, the dihemic MacA was assigned a central role. Here we have produced G. sulf urreducens MacA by recombinant expression in Escherichia coli and have solved its three-dimensional structure in three different oxidation states. Sequence comparisons group MacA into the family of diheme cytochrome c peroxidases, and the protein indeed showed hydrogen peroxide reductase activity with ABTS −2 as an electron donor. The observed K M was 38.5 ± 3.7 μM H 2 O 2 and v max was 0.78 ± 0.03 μmol of H 2 O 2 •min −1 •mg −1 , resulting in a turnover number k cat = 0.46 • s −1 . In contrast, no Fe(III) reductase activity was observed. MacA was found to display electrochemical properties similar to other bacterial diheme peroxidases, in addition to the ability to electrochemically mediate electron transfer to the soluble cytochrome PpcA. Differences in activity between CcpA and MacA can be rationalized with structural variations in one of the three loop regions, loop 2, that undergoes conformational changes during reductive activation of the enzyme. This loop is adjacent to the active site heme and forms an open loop structure rather than a more rigid helix as in CcpA. For the activation of the protein, the loop has to displace the distal ligand to the active site heme, H93, in loop 1. A H93G variant showed an unexpected formation of a helix in loop 2 and disorder in loop 1, while a M297H variant that altered the properties of the electron transfer heme abolished reductive activation.

Proteins involved in electron transfer to Fe(III) and Mn(IV) oxides by Geobacter sulfurreducens and Geobacter uraniireducens

“…However, genetic studies showed that the phenotype of the ccpA mutant strain of G. sulfurreducens was similar to that of the wild-type during growth on all tested electron acceptors (Table 5). macA (GSU0466), encoding a second peroxidase that is very similar to CcpA and capable of electron transfer to PpcA, a c-type cytochrome that is important for growth on Fe(III) citrate (Seidel et al, 2012), is upregulated only on Mn(IV) oxides (Table 4); however, a knockout of macA resulted in impaired growth on soluble and insoluble Fe(III) but not on Mn(IV) oxide (Table 5 and Fig. 1).…”

The mechanisms of extracellular electron transfer

“…This model suggests that electrons are transferred from the menoquinone pool within the inner membrane to MacA which then transfers electrons PpcA [163], which shuttles the electrons across the periplasm to a Pcc complex [33]. The Pcc complex passes electrons to the OMCs OmcE and OmcS, which may be responsible for the transfer of electrons to pili [161,165] of which OmcS is attached [14,[166][167][168], however G. sulfurreducens can also transfer electrons directly using OMCs, without the involvement of pili [165].…”

“…The deletion of macA results in a significant reduction in the capacity for Fe(III) reduction, suggesting that MacA holds an important role in iron reduction [172]. MacA is a peroxidase which is suggested to transfer electrons to the cytochrome PpcA [163]. Both cytochromes are thought to play a role as periplasmic intermediate electron transfer…”

“…constituents [163]. OmaC, a periplasmic c-type cytochrome, belongs to of one of two known Pcc protein complexes responsible for the transfer of electrons across the outer membrane [33].…”

“…Electrons are then transferred to PpcA, which facilitates electron transfer across the periplasm [161][162][163][164]. PpcA, and possibly other periplasmic cytochromes then transfer electrons to a Pcc complex (OmbB/OmbC, OmaB/OmaC and OmcB/OmcC porin-cytochrome protein complexes) that transfer the electrons through the outer membrane [33].…”