While iron is often a limiting nutrient to Biology, when the element is found in the form of heme cofactors (iron protoporphyrin IX), living systems have exceled at modifying and tailoring the chemistry of the metal. In the context of proteins and enzymes, heme cofactors are increasingly found in stoichiometries greater than one, where a single protein macromolecule contains more than one heme unit. When paired or coupled together, these protein associated heme groups perform a wide variety of tasks, such as redox communication, long range electron transfer and storage of reducing/oxidizing equivalents. Here, we review recent advances in the field of multi-heme proteins, focusing on emergent properties of these complex redox proteins, and strategies found in Nature where such proteins appear to be modular and essential components of larger biochemical pathways.

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.

Bacterial cytochrome c peroxidase (CcP) enzymes are diheme redox proteins that reduce hydrogen peroxide to water. They are canonically characterized by a peroxidatic (called L, for “low reduction potential”) active site heme, and a secondary heme (H, for “high reduction potential”) associated with electron transfer, and an enzymatic activity that exists only when the H-heme is pre-reduced to the FeII oxidation state. The pre-reduction step results in a conformational change at the active site itself, where a histidine-bearing loop will adopt an “open” conformation allowing hydrogen peroxide to bind to the FeIII of the L-heme. Notably, the enzyme from Nitrosomonas europaea does not require pre-reduction. Previously, we have shown that protein film voltammetry (PFV) is a highly useful tool in distinguishing the electrocatalytic mechanisms of the Nitromonas-type of enzyme from other CcPs. Here, we apply PFV to the recently described enzyme from Geobacter sulfurreducens, and the Geobacter S134P/V135K double mutant, which has been shown to be similar to the canonical sub-class of peroxidases and the Nitrosomonas sub-class of enzymes, respectively. Here we find that the wild-type Geobacter CcP is indeed similar electrochemically to the bacterial CcPs that require reductive activation, yet the S134P/V135K mutant shows two phases of electrocatalysis: one that is low in potential, like the wild-type enzyme, and a second, higher-potential phase that has a potential dependent upon substrate binding and pH, yet is at a potential that is very similar to the H-heme. These findings are interpreted in terms of a model where rate-limiting intra-protein electron transfer governs the catalytic performance of the S134P/V135K enzyme.

All known active forms of diheme bacterial cytochrome c peroxidase (bCcP) enzymes are described by a homodimeric state. Further the majority of bCcPs reported only display activity when the high-potential electron transfer heme of the protein (FeH) is reduced by to the ferrous oxidation state. Reduction of FeH results in a set of conformational changes allowing for the low-potential peroxidatic heme (FeL) to adopt a high-spin, five-coordinate state that is capable of binding substrate. Here we examine the impact of dimerization upon the activity of the Shewanella oneidensis bCcP by the preparation of single charge-reversal mutants at the dimer interface, and use the resulting construct to illustrate why dimerization is likely a requirement for activity in bCcPs. The E258K mutant is found to form a monomeric state in solution as characterized by size exclusion chromatography and analytical ultracentrifugation analyses. The resulting E258K monomer has a folding stability comparable to wild-type So bCcP, and an activity that is only slightly diminished (kcat/Km of 23 × 10 6 M−1 s−1). Spectroscopic and potentiometric analyses reveal that while the thermodynamic stability of the activated form of the enzyme is unchanged (characterized by the Em value of the FeHII/III couple) , the kinetic stability of the activated form of the enzyme has been greatly diminished upon generation of the monomer. Together, these data suggest a model where dimerization of bCcP enzymes is required in order to stabilize the lifetime of the activated form of the enzyme against re-oxidation of FeH and deactivation of FeL.