MauG is a novel 42 kDa diheme protein which is required for the biosynthesis of tryptophan tryptophylquinone, the prosthetic group of methylamine dehydrogenase. The visible absorption and resonance Raman spectroscopic properties of each of the two c-type hemes and the overall redox properties of MauG are described. The absorption maxima for the Soret peaks of the oxidized and reduced hemes are 403 and 418 nm for the low-spin heme and 389 and 427 nm for the high-spin heme, respectively. The resonance Raman spectrum of oxidized MauG exhibits a set of marker bands at 1503 and 1588 cm(-1) which exhibit frequencies similar to those of the nu3 and nu2 bands of c-type heme proteins with bis-histidine coordination. Another set of marker bands at 1478 and 1570 cm(-1) is characteristic of a high-spin heme. Two distinct oxidation-reduction midpoint potential (E(m)) values of -159 and -244 mV are obtained from spectrochemical titration of MauG. However, the two nu3 bands located at 1478 and 1503 cm(-1) shift together to 1467 and 1492 cm(-1), respectively, upon reduction, as do the Soret peaks of the low- and high-spin hemes in the absorption spectrum. Thus, the two hemes with distinct spectral properties are reduced and oxidized to approximately the same extent during redox titrations. This indicates that the high- and low-spin hemes have similar intrinsic E(m) values but exhibit negative redox cooperativity. After the first one-electron reduction of MauG, the electron equilibrates between hemes. This makes the second one-electron reduction of MauG more difficult. Thus, the two E(m) values do not describe redox properties of distinct hemes, but the first and second one-electron reductions of a diheme system with two equivalent hemes. The structural and mechanistic implications of these findings are discussed.

Surface-enhanced resonance Raman scattering (SERRS) spectra of biological species are often different from their resonance Raman (RR) spectra. A home-designed Raman flow system is used to determine the factors that contribute to the difference between the SERRS and RR of met-myoglobin (metMb). The results indicate that both the degree of protein-nanoparticles interaction and the laser irradiation contribute to the structural changes and are responsible for the observed differences between the SERRS and RR spectra of metMb. The prolonged adsorption of the protein molecules on the nanoparticle surface, which is the condition normally used for the conventional SERRS experiments, disturbs the heme pocket structure and facilitates the charge transfer process and the photoinduced transformation of proteins. The disruption of the heme pocket results in the loss of the distal water molecule, and the resulting SERRS spectrum of metMb shows a 5-coordinated high-spin heme. The flow system, when operated at a moderately high flow rate, can basically eliminate the factors that disturb the protein structure while maintaining a high enhancement factor. The SERRS spectrum obtained from a 1 x 10 (-7) M metMb solution using this flow system is basically identical to the RR spectrum of a 5 x 10 (-4) M metMb solution. Therefore, the Raman flow system reported here should be useful for characterizing the protein-nanoparticles interaction and the native structure of proteins using SERRS spectroscopy.

The diheme enzyme MauG catalyzes the posttranslational modification of a precursor protein of methylamine dehydrogenase (preMADH) to complete the biosynthesis of its protein-derived tryptophan tryptophylquinone (TTQ) cofactor. It catalyzes three sequential two-electron oxidation reactions which proceed through a high valent bis-Fe(IV) redox state. Tyr294, the unusual distal axial ligand of one c-type heme, was mutated to His and the crystal structure of Y294H MauG in complex with preMADH reveals that this heme now has His-His axial ligation. Y294H MauG is able to interact with preMADH and participate in inter-protein electron transfer, but it is unable to catalyze the TTQ biosynthesis reactions that require the bis-Fe(IV) state. This mutation not only affects the redox properties of the six-coordinate heme but also the redox and CO-binding properties of the five-coordinate heme, despite the 21 Å separation of the heme iron centers. This highlights the communication between the hemes which in wild-type MauG behave as a single diheme unit. Spectroscopic data suggest that Y294H MauG can stabilize a high valent redox state equivalent to Fe(V), but it appears to be an Fe(IV)=O/π radical at the five-coordinate heme rather than the bis-Fe(IV) state. This compound I-like intermediate does not catalyze TTQ biosynthesis, demonstrating that the bis-Fe(IV) state, which is stabilized by Tyr294, is specifically required for this reaction. The TTQ biosynthetic reactions catalyzed by wild-type MauG do not occur via direct contact with the Fe(IV)=O heme, but via long range electron transfer through the six-coordinate heme. Thus, a critical feature of the bis-Fe(IV) species may be that it shortens the electron transfer distance from preMADH to a high valent heme iron.

The effect of an external magnetic field on the intensity of electrogenerated chemiluminescence (eel) was determined for nine systems involving anthracene, 9,10-diphenylanthracene (DPA), rubrene, 1,3,6,8-tetraphenylpyrene (TPP), and fluoranthene as emitting species in A.A-dimethylformamide (DMF) solutions. Enhancements in emission intensity up to 27 % with increasing field strength were noted for the energy-deficient oxidations of anthracene, DPA, rubrene, and TPP anion radicals by Wurster's Blue cation, for the energy-deficient oxidation of fluoranthene anion radical by the cation radical of 10-methylphenothiazine, and for the energy-deficient reduction of the rubrene cation radical by the anion radical derived from /j-benzoquinone. No field effect was seen on luminescence arising from the apparently energy-sufficient mutual annihilation of the anion and cation radicals derived from DPA. The field enhanced luminescence from the reaction between the rubrene anion and cation radicals, but it exerted no effect on the intensity of emission from the TPP anion-cation radical annihilation. These results have been interpreted as reflecting a dual mechanism for chemiluminescent electron-transfer processes which divides on or about the line of energy sufficiency. In particular it is suggested that the field effect accompanying luminescence from energy-deficient systems arises from an inhibition by the field of the rate of quenching of emitter triplets by radical ions. Thus, the results are consistent with the triplet mechanism for luminescence from energydeficient systems. This interpretation also indicates that energy-sufficient systems yield luminescence without required triplet intermediates. For the two marginal systems involving rubrene and TPP alone, it is suggested that the rubrene anion-cation annihilation gives rise to luminescence predominantly via the triplet mechanism, while the TPP anion-cation reaction may be essentially energy sufficient, and that most luminescence from this process arises by direct population of the emitting state.(2) K.