Search

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 excelled 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. This article is part of a Special Issue entitled: Metals in Bioenergetics and Biomimetics Systems., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Protein-protein interactions are well-known to regulate enzyme activity in cell signaling and metabolism. Here, we show that protein-protein interactions regulate the activity of a respiratory-chain enzyme, CymA, by changing the direction or bias of catalysis. CymA, a member of the widespread NapC/NirT superfamily, is a menaquinol-7 (MQ-7) dehydrogenase that donates electrons to several distinct terminal reductases in the versatile respiratory network of Shewanella oneidensis . We report the incorporation of CymA within solid-supported membranes that mimic the inner membrane architecture of S. oneidensis . Quartz-crystal microbalance with dissipation (QCM-D) resolved the formation of a stable complex between CymA and one of its native redox partners, flavocytochrome c3 (Fcc3) fumarate reductase. Cyclic voltammetry revealed that CymA alone could only reduce MQ-7, while the CymA-Fcc3 complex catalyzed the reaction required to support anaerobic respiration, the oxidation of MQ-7. We propose that MQ-7 oxidation in CymA is limited by electron transfer to the hemes and that complex formation with Fcc3 facilitates the electron-transfer rate along the heme redox chain. These results reveal a yet unexplored mechanism by which bacteria can regulate multibranched respiratory networks through protein-protein interactions.

Shewanella oneidensis MR-1 has the ability to use many external terminal electron acceptors during anaerobic respiration, such as DMSO. The pathway that facilitates this electron transfer includes the decahaem cytochrome DmsE, a paralogue of the MtrA family of decahaem cytochromes. Although both DmsE and MtrA are decahaem cytochromes implicated in the long-range electron transfer across a ~300 Å (1 Å=0.1 nm) wide periplasmic 'gap', MtrA has been shown to be only 105 Å in maximal length. In the present paper, DmsE is further characterized via protein film voltammetry, revealing that the electrochemistry of the DmsE haem cofactors display macroscopic potentials lower than those of MtrA by 100 mV. It is possible this tuning of the redox potential of DmsE is required to shuttle electrons to the outer-membrane proteins specific to DMSO reduction. Other decahaem cytochromes found in S. oneidensis, such as the outer-membrane proteins MtrC, MtrF and OmcA, have been shown to have electrochemical properties similar to those of MtrA, yet possess a different evolutionary relationship.

The potential exploitation of metal-reducing bacteria as a means for environmental cleanup or alternative fuel is an exciting prospect; however, the cellular processes that would allow for these applications need to be better understood. MtrA is a periplasmic decaheme c-type cytochrome from Shewanella oneidensis involved in the reduction of extracellular iron oxides and therefore is a critical element in Shewanella ability to engage in extracellular charge transfer. As a relatively small 333-residue protein, the heme content is surprisingly high. MtrA is believed to obtain electrons from the inner membrane-bound quinol oxidoreductase, CymA, and shuttle them across the outer membrane to MtrC, another decaheme cytochrome that directly interacts with insoluble metal oxides. How MtrA is able to perform this task is a question of interest. Here through the use of two solution-based techniques, small-angle X-ray scattering (SAXS) and analytical ultracentrifugation (AUC), we present the first structural analysis of MtrA. Our results establish that between 0.5 and 4 mg/mL, MtrA exists as a monomeric protein that is shaped like an extended molecular "wire" with a maximum protein dimension (D(max)) of 104 Å and a rod-like aspect ratio of 2.2 to 2.5. This study contributes to a greater understanding of how MtrA fulfills its role in the redox processes that must occur before electrons reach the outside of the cell., (© 2011 American Chemical Society)

Examining electron transfer between two proteins with identical spectroscopic signatures is a challenging task. It is supposed that several multiheme cytochromes in Shewanella oneidensis form a molecular "wire" through which electrons are transported across the cellular space and a direct study of this transient protein-protein interaction has not yet been reported. In this study, we present variations on catalytic protein film voltammetry and an anaerobic affinity chromatography assay to demonstrate unidirectional electron transfer between proposed protein pairs. Through use of these techniques, we are able to confirm the transient interactions between these cytochromes, supporting the model of electron transfer that is present in the literature.