A molecular electrocatalyst is reported that reduces protons to hydrogen (H2) in neutral water under aerobic conditions. The biomolecular catalyst is made from cobalt substitution of microperoxidase-11, a water-soluble heme-undecapeptide derived from the protein horse cytochrome c. In aqueous solution at pH 7.0, the catalyst operates with near quantitative Faradaic efficiency, a turnover frequency ~6.7 s(-1) measured over 10 min at an overpotential of 852 mV, and a turnover number of 2.5 × 10(4). Catalyst activity has low sensitivity to oxygen. The results show promise as a hydrogenase functional mimic derived from a biomolecule.

Significance To probe the effect of heme ruffling on electron transport, we studied three cytochromes that display wide variation in the heme ruffling distortion. Ruffling is characterized by a low-frequency heme mode in the region 45–60 cm −1 and by a photoreduction cross-section that displays strong variation as a function of the magnitude of the distortion. Given the similarity in the distance between the heme and the nearest aromatic amino acid for all three proteins, the order-of-magnitude changes in photoreduction rate demonstrate that the ruffling coordinate can serve as a control mechanism for electron transport in heme proteins. Major differences in heme ruffling are noted for cytochrome c when bound to the mitochondrial membrane compared with its solution structure.

Hemes are ubiquitous in biology and carry out a wide range of functions. The heme group is largely invariant across proteins with different functions, although there are a few variations seen in nature. The most common variant is heme c, which is formed by a post-translational modification in which heme is covalently linked to two Cys residues on the polypeptide via thioether bonds. In this Account, the influence of this covalent attachment on heme c properties and function is discussed, and examples of how covalent attachment has been used in selected applications are presented. Proteins that bind heme c are among the most well-characterized proteins in biochemistry. Most of these proteins are cytochromes c (cyts c) that serve as electron carriers in photosynthesis and respiration. Despite the intense study of cyts c, the functional significance of heme covalent attachment has remained elusive. One observation is that heme c reaches a lower reduction potential in nature than its noncovalently linked counterpart, heme b, when comparing proteins with the same axial ligands. Furthermore, covalent attachment is known to enhance protein stability and allow the heme to be relatively solvent exposed. However, an inorganic chemistry perspective on the effects of covalent attachment has been lacking. Spectroscopic measurements and computations on cyts c and model systems reveal a number of effects of covalent attachment on heme electronic structure and reactivity. One is that the predominant nonplanar ruffling distortion seen in heme c lowers heme reduction potential. Another is that covalent attachment influences the interaction of the heme iron with the proximal His ligand. Heme ruffling also has been shown to influence electronic coupling to redox partners and, therefore, electron transfer rates by altering the distribution of the orbital hole on the porphyrin in oxidized cyt c. Another consequence of heme covalent attachment is the strong vibrational coupling seen between the iron and the protein surface as revealed by nuclear resonance vibrational spectroscopy studies. Finally, heme covalent attachment is proposed to be an important feature supporting multiple roles of cyt c in programmed cell death (apoptosis). Heme covalent attachment is not only vital for the biological functions of cyt c but also provides a useful handle in a number of applications. For one, the engineering of heme c onto an exposed portion of a protein of interest has been shown to provide a visible affinity purification tag. In addition, peptides with covalently attached heme, known as microperoxidases, have been studied as model compounds and oxidation catalysts and, more recently, in applications for energy conversion and storage. The wealth of insight gained about heme c through fundamental studies of cyts c forms a basis for future efforts toward engineering natural and artificial cytochromes for a variety of applications.

The active site of cytochrome c (Cyt c ) consists of a heme covalently linked to a pentapeptide segment (Cys-X-X-Cys-His), which provides a link between the heme and the protein surface, where the redox partners of Cyt c bind. To elucidate the vibrational properties of heme c , nuclear resonance vibrational spectroscopy (NRVS) measurements were performed on 57 Fe-labeled ferric Hydrogenobacter thermophilus cytochrome c 552 , including 13 C 8 -heme–, 13 C 5 15 N-Met–, and 13 C 15 N-polypeptide (pp)–labeled samples, revealing heme-based vibrational modes in the 200- to 450-cm −1 spectral region. Simulations of the NRVS spectra of H. thermophilus cytochrome c 552 allowed for a complete assignment of the Fe vibrational spectrum of the protein-bound heme, as well as the quantitative determination of the amount of mixing between local heme vibrations and pp modes from the Cys-X-X-Cys-His motif. These results provide the basis to propose that heme-pp vibrational dynamic couplings play a role in electron transfer (ET) by coupling vibrations of the heme directly to vibrations of the pp at the protein–protein interface. This could allow for the direct transduction of the thermal (vibrational) energy from the protein surface to the heme that is released on protein/protein complex formation, or it could modulate the heme vibrations in the protein/protein complex to minimize reorganization energy. Both mechanisms lower energy barriers for ET. Notably, the conformation of the distal Met side chain is fine-tuned in the protein to localize heme-pp mixed vibrations within the 250- to 400-cm −1 spectral region. These findings point to a particular orientation of the distal Met that maximizes ET.