Characterization of electron tunneling and hole hopping reactions between different forms of MauG and methylamine dehydrogenase within a natural protein complex.

Long range electron transfer (ET) is a fundamental process to the fields of chemistry, biology and physics. Two different mechanisms for long range ET have been considered. One is direct electron tunneling which occurs in a single step reaction from electron donor to acceptor (1–5), and the other one is multi-step hopping (6). To fully describe the relevance of these processes to biology, two key questions remain to be answered. How does ET occur over large distances through and between proteins with speed and specificity? How does the protein matrix and nature of the redox centers determine contributions of electron tunneling and hole hopping to the overall mechanism of ET? This study determines, analyzes and compares the temperature dependencies of the rates of two different ET reactions that occur between the diheme enzyme MauG (7) and different forms of methylamine dehydrogenase (MADH) (8), which bears the tryptophan tryptophylquinone (TTQ) redox cofactor. One reaction is ET from diferrous MauG to the quinone form of MADH. The other is ET from a biosynthetic precursor of MADH (preMADH) (9) to a high valent bis-Fe(IV) form (10) of MauG. The results demonstrate that the former reaction occurs via single-step electron tunneling while the latter reaction occurs via multi-step hole hopping. During long range electron tunneling through proteins, the amino acid residues that mediate ET do not undergo a change in redox state. They are simply a conductive matrix. In contrast, during hopping certain intervening amino acid residues are reversibly oxidized and reduced and serve as intermediate staging points or stepping stones for the hops. Two alternative hopping mechanisms are possible. If the electron donor reduces the intermediate which in turn reduces the electron acceptor, then this would be electron hopping. If the electron acceptor oxidizes the intermediate which in turn oxidizes the electron donor, then this would be hole hopping. Hopping has been described in non-biological systems such as organic semiconductors which have been described as hole charge transporters (11) and through molecular wires (12). In biological systems, hole hopping through DNA has been demonstrated (13–17). The purine base guanine is the preferred hopping relay as it the easiest base to oxidize. The efficiency of DNA as a charge carrier depends in large part on the spacing between guanine bases. While it is not likely relevant to metabolism, hopping does allow rapid charge transfer through DNA. Documentation of hopping through proteins has been difficult to achieve and evidence for this mechanism has only been obtained for a few systems, ribonucleotide reductase (18, 19), DNA photolyase (20), an engineered ruthenium labeled azurin (21) and cytochrome c peroxidase (22). A recent sitedirected mutagenesis study of tryptophan 199 of MauG suggested that this residue mediates hole hopping during the long range ET that is required for MauG-dependent TTQ biosynthesis (23). Thus, the MauG system provides a rare opportunity to characterize a natural physiologically relevant hopping mechanism in a protein system. MauG is a diheme enzyme that catalyzes the final steps in the biosynthesis of the protein-derived cofactor, TTQ in MADH (24–26). The biosynthetic process is a six-electron oxidation of the precursor protein, preMADH, which possesses monohydroxylated residue βTrp57 (Figure 1A). The completion of TTQ biosynthesis requires that βTrp57 is covalently crosslinked to βTrp108, a second oxygen atom is inserted into the indole ring of βTrp57, and the quinol species is oxidized to the quinone (9). These reactions require the formation of a high valent bis-Fe(IV) intermediate of MauG in which one heme is present as Fe(IV)=O with an axial His ligand and the other is Fe(IV) with no exogenous ligand (10) and axial ligands provided by His and Tyr residues (24) (Figure 1B). Significantly, the crystal structure of the MauG-preMADH complex revealed that βTrp57 and βTrp108 do not make direct contact with either heme of MauG (24) (Figure 1C). In fact, the distance between the side-chain of βTrp108 of preMADH and the iron of the oxygen-binding five-coordinate heme is 40.1 A, and the closest distance from the residues of preMADH that are modified to the iron of the six-coordinate heme is 19.4 A. Nonetheless, addition of H2O2 to MauG-preMADH crystals causes synthesis of the mature TTQ cofactor in crystallo demonstrating that the orientation of the proteins in the crystallized complex is catalytically active (24). Thus, these catalytic reactions absolutely require long range ET. This study applies Marcus theory (3) to analyze the temperature dependencies of the rates of two different ET reactions that occur between these proteins (Figure 2). One is a non-biosynthetic ET reaction from diferrous MauG to the quinone form of MADH (27). The other is a biosynthetic ET reaction from preMADH to the bis-Fe(IV) form of MauG (28). The results are consistent with the former reaction occurring via single-step electron tunneling while the latter reaction occurring via multi-step hole hopping. Figure 1 A. MauG catalyzes the conversion of monohydroxylated βTrp57 and βTrp108 of preMADH to TTQ. [O] is oxidation equivalents. B. The bis-Fe(IV) state of MauG is formed by reaction of [O] with diferric MauG. The residues which form the axial ... Figure 2 Schematic depiction of the electron transfer reactions (A) from diferrous MauG to quinone MADH and (B) from preMADH to the nearest heme of bis-Fe(IV) MauG.