The nuclear genome of the model organism Chlamydomonas reinhardtii contains genes for a dozen hemoglobins of the truncated lineage. Of those, THB1 is known to be expressed, but the product and its function have not yet been characterized. We present mutagenesis, optical, and nuclear magnetic resonance data for the recombinant protein and show that at pH near neutral in the absence of added ligand, THB1 coordinates the heme iron with the canonical proximal histidine and a distal lysine. In the cyanomet state, THB1 is structurally similar to other known truncated hemoglobins, particularly the heme domain of Chlamydomonas eugametos LI637, a light-induced chloroplastic hemoglobin. Recombinant THB1 is capable of binding nitric oxide (NO•) in either the ferric or ferrous state and has efficient NO• dioxygenase activity. By using different C. reinhardtii strains and growth conditions, we demonstrate that the expression of THB1 is under the control of the NIT2 regulatory gene and that the hemoglobin is linked to the nitrogen assimilation pathway.

Iron-protoporphyrin IX, or b heme, is utilized as such by a large number of proteins and enzymes. In some cases, notably the c-type cytochromes, this group undergoes a posttranslational covalent attachment to the polypeptide chain, which adjusts the physicochemical properties of the holoprotein. The hemoglobin from the cyanobacterium Synechocystis sp. PCC 6803 (GlbN), contrary to the archetypical hemoglobin, modifies its b heme covalently. The posttranslational modification links His117, a residue that does not coordinate the iron, to the porphyrin 2-vinyl substituent and forms a hybrid b/c heme. The reaction is an electrophilic addition that occurs spontaneously in the ferrous state of the protein. This apparently facile type of heme modification has been observed in only two cyanobacterial GlbNs. To explore the determinants of the reaction, we examined the behavior of Synechocystis GlbN variants containing a histidine at position 79, which is buried against the porphyrin 4-vinyl substituent. We found that L79H/H117A GlbN bound the heme weakly but nevertheless formed a cross-link between His79 Nε2 and the heme 4-Cα. In addition to this linkage, the single variant L79H GlbN also formed the native His117-2-Cα bond yielding an unprecedented bis-alkylated protein adduct. The ability to engineer the doubly modified protein indicates that the histidine-heme modification in GlbN is robust and could be engineered in different local environments. The rarity of the histidine linkage in natural proteins, despite the ease of reaction, is proposed to stem from multiple sources of negative selection.

This paper presents results from a pulsed-laser cavity ring-down spectrometer with novel field programable gate array real-time data collection. We show both theoretically and experimentally that the data extraction can be achieved from a single cavity ringdown event, and that the absorbance can be determined without the need to fit the ringdown time explicitly. This methodology could potentially provide data acquisition rate up to 1 MHz, with the accuracy and precision comparable to nonlinear least squares fitting algorithms.

Bacterial diheme cytochrome c peroxidases form part of the detoxification pathways in the periplasmic space that convert hydrogen peroxide to water. The turnover of peroxide requires a two-electron reduction, and these reducing equivalents must be delivered to the peroxidase by soluble electron transfer proteins. This study focuses on the electron transfer complexes formed between the diheme cytochrome c peroxidase from Shewanella oneidensis (So CcP) and its native electron donor ScyA. Due to the transient nature of this interaction and its presumed dependence on redox state of the two binding partners, traditional biophysical analysis of this system has proved challenging. Here, we report the use of RosettaDock to predict the protein-protein interaction surfaces on So CcP when in the inactive, oxidized state and the active, semi-reduced state. We validate these models by generating site-directed mutants designed to interfere with specific hydrophobic interfaces suggested in the docking analysis and measuring their kinetic competence for both single-electron reductive activation and two-electron peroxide turnover. These results suggest how redox-driven changes in So CcP conformation regulate protein-protein interactions.