Neuroglobin (Ngb) is a recently discovered vertebrate heme protein that can reversibly bind oxygen that is expressed in the brain. Zebratish and human Ngb share about 50% amino acid sequence identity. These Ngb proteins consist of four compact protein structural unit "modules" referred to as M1-M4. In the present study, we investigated the effects of module substitution on the properties of Ngb. Specifically, we prepared and characterized a chimeric ZHZZ Ngb in which the heme-binding module M2 of zebrafish Ngb was replaced by the comparable human Ngb module. Our results showed that the chimeric ZHZZ was stable and formed almost the identical heme-environmental and alpha-helical Structure as the human and zebrafish Ngb proteins, suggesting that the structure of Ngb has been evolutionarily conserved. (c) 2005 Elsevier Inc. All rights reserved.