YddV from Escherichia coli (Ec) is a novel globin-coupled heme-based oxygen sensor protein displaying diguanylate cyclase activity in response to oxygen availability. In this study, we quantified the turnover numbers of the active [Fe(III), 0.066 min(-1); Fe(II)-O(2) and Fe(II)-CO, 0.022 min(-1)] [Fe(III), Fe(III)-protoporphyrin IX complex; Fe(II), Fe(II)-protoporphyrin IX complex] and inactive forms [Fe(II) and Fe(II)-NO, <0.01 min(-1)] of YddV for the first time. Our data indicate that the YddV reaction is the rate-determining step for two consecutive reactions coupled with phosphodiesterase Ec DOS activity on cyclic di-GMP (c-di-GMP) [turnover number of Ec DOS-Fe(II)-O(2), 61 min(-1)]. Thus, O(2) binding and the heme redox switch of YddV appear to be critical factors in the regulation of c-di-GMP homeostasis. The redox potential and autoxidation rate of heme of the isolated heme domain of YddV (YddV-heme) were determined to be -17 mV versus the standard hydrogen electrode and 0.0076 min(-1), respectively. The Fe(II) complexes of Y43A and Y43L mutant proteins (residues at the heme distal side of the isolated heme-bound globin domain of YddV) exhibited very low O(2) affinities, and thus, their Fe(II)-O(2) complexes were not detected on the spectra. The O(2) dissociation rate constant of the Y43W protein was >150 s(-1), which is significantly larger than that of the wild-type protein (22 s(-1)). The autoxidation rate constants of the Y43F and Y43W mutant proteins were 0.069 and 0.12 min(-1), respectively, which are also markedly higher than that of the wild-type protein. The resonance Raman frequencies representing ν(Fe-O(2)) (559 cm(-1)) of the Fe(II)-O(2) complex and ν(Fe-CO) (505 cm(-1)) of the Fe(II)-CO complex of Y43F differed from those (ν(Fe-O(2)), 565 cm(-1); ν(Fe-CO), 495 cm(-1)) of the wild-type protein, suggesting that Tyr43 forms hydrogen bonds with both O(2) and CO molecules. On the basis of the results, we suggest that Tyr43 located at the heme distal side is important for the O(2) recognition and stability of the Fe(II)-O(2) complex, because the hydroxyl group of the residue appears to interact electrostatically with the O(2) molecule bound to the Fe(II) complex in YddV. Our findings clearly support a role of Tyr in oxygen sensing, and thus modulation of overall conversion from GTP to pGpG via c-di-GMP catalyzed by YddV and Ec DOS, which may be applicable to other globin-coupled oxygen sensor enzymes.

We analyzed the vibrational frequencies of the Fe-bound carbon monoxide (CO) of myoglobin reconstituted with a series of chemically modified heme cofactors possessing a heme Fe atom with a variety of electron densities. The study revealed that the stretching frequency of Fe-bound CO (ν(CO)) increases with decreasing electron density of the heme Fe atom (ρ(Fe)). This finding demonstrated that the ν(CO) value can be used as a sensitive measure of the ρ(Fe) value and that the π back-donation of the heme Fe atom to CO is affected by the heme π-system perturbation induced through peripheral side chain modifications.

Reliable sample delivery is essential to biological imaging using X-ray Free Electron Lasers (XFELs). Continuous injection using the Gas Dynamic Virtual Nozzle (GDVN) has proven valuable, particularly for time-resolved studies. However, many important aspects of GDVN functionality have yet to be thoroughly understood and/or refined due to fabrication limitations. We report the application of 2-photon polymerization as a form of high-resolution 3D printing to fabricate high-fidelity GDVNs with submicron resolution. This technique allows rapid prototyping of a wide range of different types of nozzles from standard CAD drawings and optimization of crucial dimensions for optimal performance. Three nozzles were tested with pure water to determine general nozzle performance and reproducibility, with nearly reproducible off-axis jetting being the result. X-ray tomography and index matching were successfully used to evaluate the interior nozzle structures and identify the cause of off-axis jetting. Subsequent refinements to fabrication resulted in straight jetting. A performance test of printed nozzles at an XFEL provided high quality femtosecond diffraction patterns.

Cytochrome c oxidase (CcO) reduces dioxygen to water and harnesses the chemical energy to drive proton translocation across the inner mitochondrial membrane by an unresolved mechanism. By using time-resolved serial femtosecond crystallography, we identified a key oxygen intermediate of bovine CcO. It is assigned to the PR-intermediate, which is characterized by specific redox states of the metal centers and a distinct protein conformation. The heme a3 iron atom is in a ferryl (Fe4+ = O2−) configuration, and heme a and CuB are oxidized while CuA is reduced. A Helix-X segment is poised in an open conformational state; the heme a farnesyl sidechain is H-bonded to S382, and loop-I-II adopts a distinct structure. These data offer insights into the mechanism by which the oxygen chemistry is coupled to unidirectional proton translocation.

We analyzed the oxygen (O2) and carbon monoxide (CO) binding properties of the H64L mutant of myoglobin reconstituted with chemically modified heme cofactors possessing a heme Fe atom with a variety of electron densities, in order to elucidate the effect of the removal of the distal His64 on the control of both the O2 affinity and discrimination between O2 and CO of the protein by the intrinsic heme Fe reactivity through the electron density of the heme Fe atom (ρFe). The study revealed that, as in the case of the native protein, the O2 affinity of the H64L mutant protein is regulated by the ρFe value in such a manner that the O2 affinity of the protein decreases, due to an increase in the O2 dissociation rate constant, with a decrease in the ρFe value, and that the O2 affinities of the mutant and native proteins are affected comparably by a given change in the ρFe value. On the other hand, the CO affinity of the H64L mutant protein was found to increase, due to a decrease in the CO dissociation rate constant, with a decrease in the ρFe value, whereas that of the native protein was essentially independent of a change in the ρFe value. As a result, the regulation of the O2/CO discrimination in the protein through the ρFe value is affected by the distal His64. Thus, the study revealed that the electronic tuning of the intrinsic heme Fe reactivity through the ρFe value plays a vital role in the regulation of the protein function, as the heme environment furnished by the distal His64 does.