Iron Fe is an essential element for all of lives. Due to its wide range of redox potential and ability of coordination with small molecules, the iron is involved in many important biological processes such as energy, materials and signal conversions. The iron is acquired from dietary foods into cells, where some proteins work in absorption, transport, sensing, storage and utilization of the iron, i.e., iron dynamics in biological systems. Professor Yoshitsugu Shiro has been interested in proteins/enzymes related to such biological irons, and has investigated their functions in molecular/atomic levels. His major achievements are summarized below.

1. NO dynamics in cells based on nitric oxide reductase
Nitric oxide NO is generated in denitrification, a kind of anaerobic respiration of microorganism, but, due to its radical character, it exhibits high cytotoxicity. In the denitrification, NO generated by nitrite reductase (NiR: NO₂^- + 2H⁺ + e^- → NO + H₂O) is reduced to nitrous oxide N₂O for the NO detoxification by nitric oxide reductases (NOR: 2NO + 2H⁺ + 2e^- → N₂O + H₂O), which are iron-containing enzymes. Prof. Shiro reported the first crystal structure NOR, and proposed the molecular mechanism of the NO reduction reaction based on its structure. Most recently, he successfully characterized the coordination and electronic structure of the iron-bound NO in the active site of the short-lived intermediates, which are transiently appeared in the NOR enzymatic reaction, by using time-resolved spectroscopic and crystallographic techniques. In addition, he also determined the mechanism how NO is transfer from NiR to NOR without being diffused inside the cell. His achievement on NOR has been paid much attention in not only chemistry and biochemistry fields, but also basic biology. This is because bacterial NOR shares the same ancestor as cytochrome oxidases, aerobic (O₂-utilizing) respiratory enzymes. The knowledge on NOR has been stimulating discussion on molecular evolution of the respiratory enzymes. In addition, since N₂O, product of the NOR reaction, plays as ozone-depleting gas as well as green house gas in the global level, environmental sciences have interest in his achievement.

2. Evaluation of oxygenase reactions in molecular level
Oxygenation reaction is an important biological process, in which atomic oxygen(s) of O₂ are directly incorporated into substrates. The reaction(s) are catalyzed by enzymes, the so-called oxygenase(s), and are involved in synthesis and metabolism of a variety of bio-compounds. Indoleamine dioxygenase IDO is a heme (iron-porphyrin) containing enzyme, and a key player of the tryptophan metabolism in mammal. Prof. Shiro reported the first crystal structure of human IDO and proposed its molecular mechanism of the enzymatic reaction. Since IDO is a drug-target, he collaborated to develop and elucidate the IDO inhibitors with organic chemists of universities and pharmaceutical companies. Cytochrome P450 is another type of the heme containing oxygenase, but Prof. Shiro had interest in a novel P450 enzyme which utilizes hydrogen peroxide H₂O₂ in place of O₂, protons and electrons in its oxygenation reaction. His structural and functional studies on this novel P450 enzyme stimulated organic chemists who endeavor to develop novel bio-catalysis in industrial usage, because H₂O₂ is an inexpensive oxidant.

3. Studies on intra- and inter-molecular signal transduction in oxygen sensor
As stated above, O₂ is a key gas molecule in life cycle of all living system. So, the cells monitor the O₂ concentration by O₂-sensor proteins for their survival. The O₂ sensor protein system of Rhizobium in root nodule regulates expression of the nitrogen-fixation enzyme, nitrogenase, in respond to the O₂ concentration. Association or dissociation of O₂ to or from the heme iron in the sensor protein acts as a trigger of the sensing. Prof. Shiro gave a lot of structural and functional information of such O₂ triggering in the sensor protein, which were revealed by a variety of experimental techniques. His studies contributed to stimulation of studies on metal-containing gas sensor proteins.

4. Structural and functional studies of proteins related to iron dynamics
In human, the iron in dietary foods is absorbed in duodenum. In this process, ferric iron Fe³⁺ in diet is reduced to ferrous iron Fe²⁺ with ascorbate by the ferric reductase Dcytb, and then transported into the duodenal cell across the cellular membrane with DMT1 (divalent metal transporter). Prof. Shiro determined the crystal structure of Dcytb, and revealed the ferric iron and ascorbate binding site, that provided insight into the intramolecular electron transfer across the cellular membrane. Currently, based on the molecular science, his group developed a model cell system to investigate the iron absorption of duodenal cell, in collaboration with cell biologists. On the other hand, pathogenic bacteria acquire the iron from of heme of the hemoglobin in red blood cell of host. In this heme acquisition, some proteins such as heme importer, heme exporter, heme-sensor protein and heme oxygenase work. Structures of all these proteins were crystallographically determined by his group. Among these proteins, the transporters (DMT1, heme-exporter and heme importer) dynamically work by using the biological energy such as ATP and the proton gradient, so that these studies will be developed in the dynamic structural characterization of proteins.

As highlighted above, Prof. Shiro has provided new insights into structures and functions of iron-related proteins/enzymes, and has highly contributed to advancement of biological inorganic chemistry field. Also, his contribution would be extended to "cell biology of metals", e.g., the iron dynamics in biological system will be systematically examined in cellular level on the basis of his molecular information. These studies would provide bases to organize and to manage the new chemical field "Integrated Biometal Science (IBmS)". Therefore, his achievement has been recognized as worthy of the Chemical Society of Japan (CSJ) Award.