The diheme enzyme MauG catalyzes posttranslational modifications of a methylamine dehydrogenase precursor protein to generate a tryptophan tryptophylquinone cofactor. The MauG-catalyzed reaction proceeds via a bis -Fe(IV) intermediate in which one heme is present as Fe(IV)=O and the other as Fe(IV) with axial histidine and tyrosine ligation. Herein, a unique near-infrared absorption feature exhibited specifically in bis -Fe(IV) MauG is described, and evidence is presented that it results from a charge-resonance-transition phenomenon. As the two hemes are physically separated by 14.5 Å, a hole-hopping mechanism is proposed in which a tryptophan residue located between the hemes is reversibly oxidized and reduced to increase the effective electronic coupling element and enhance the rate of reversible electron transfer between the hemes in bis -Fe(IV) MauG. Analysis of the MauG structure reveals that electron transfer via this mechanism is rapid enough to enable a charge-resonance stabilization of the bis -Fe(IV) state without direct contact between the hemes. The finding of the charge-resonance-transition phenomenon explains why the bis -Fe(IV) intermediate is stabilized in MauG and does not permanently oxidize its own aromatic residues.

The diheme enzyme MauG catalyzes the posttranslational modification of the precursor protein of methylamine dehydrogenase (preMADH) to complete biosynthesis of its protein-derived tryptophan tryptophylquinone (TTQ) cofactor. Catalysis proceeds through a high valent bis-Fe(IV) redox state and requires long-range electron transfer (ET), as the distance between the modified residues of pre-MADH and the nearest heme iron of MauG is 19.4 Å. Trp199 of MauG resides at the MauG-preMADH interface, positioned midway between the residues that are modified and the nearest heme. W199F and W199K mutations did not affect the spectroscopic and redox properties of MauG, or its ability to stabilize the bis-Fe(IV) state. Crystal structures of complexes of W199F/K MauG with pre-MADH showed no significant perturbation of the MauG-preMADH structure or protein interface. However, neither MauG variant was able to synthesize TTQ from preMADH. In contrast, an ET reaction from diferrous MauG to quinone MADH, which does not require the bis-Fe(IV) intermediate, was minimally affected by the W199F/K mutations. W199F/K MauGs were able to oxidize quinol MADH to form TTQ, the putative final two-electron oxidation of the biosynthetic process, but with k cat ∕K m values approximately 10% that of wild-type MauG. The differential effects of the W199F/K mutations on these three different reactions are explained by a critical role for Trp199 in mediating multistep hopping from preMADH to bis-Fe(IV) MauG during the long-range ET that is required for TTQ biosynthesis.cytochrome | electron hopping | peroxidase | protein oxidation | protein radical L ong-range electron transfer (ET) through proteins is required for biological processes including respiration, photosynthesis, and metabolism. Mechanisms by which ET occurs over large distances to specific sites within a protein have been extensively studied (1-4). For interprotein ET, kinetic mechanisms are more complex, as the overall redox reaction requires additional steps such as protein-protein association and reorientation of the protein complex to optimize the system for ET (5, 6). "Long-range catalysis" is a related process in which the redox center that provides the oxidizing or reducing power is physically distinct from the site of chemical reaction of the substrate, so that long-range ET is required for catalysis. Thus far two enzymes have been postulated to employ long-range catalysis. Ribonucleotide reductase (RNR) catalyzes the formation of deoxyribonucleotides from ribonucleotides by long-range ET via multiple tyrosyl residues (7,8). DNA photolyase is a flavoprotein that catalyzes DNA repair of pyrimidine-pyrimidine dimers via multiple tryptophan residues (9). In these enzymes it is believed that the long-range ET proceeds by hopping (10) through residues that can stabilize a radical state, rather than via a single long-range electron tunneling event.

Plants synthesize carotenoids essential for plant development and survival. These metabolites also serve as essential nutrients for human health. The biosynthetic pathway leading to all plant carotenoids occurs in chloroplasts and other plastids and requires 15-cis-ζ-carotene isomerase (Z-ISO). It was not certain whether isomerization was achieved by Z-ISO alone or in combination with other enzymes. Here we show that Z-ISO is a bona fide enzyme and integral membrane protein. Z-ISO independently catalyzes the cis-to-trans isomerization of the 15–15′ C=C bond in 9,15,9′-cis-ζ-carotene to produce the substrate required by the following biosynthetic pathway enzyme. We discovered that isomerization depends upon a ferrous heme b cofactor that undergoes redox-regulated ligand-switching between the heme iron and alternate Z-ISO amino acid residues. Heme b-dependent isomerization of a large, hydrophobic compound in a membrane is unprecedented. As an isomerase, Z-ISO represents a new prototype for heme b proteins and potentially utilizes a novel chemical mechanism.