H-2 production under aerobic conditions has been proposed as an alternative method to overcome the fundamentally low yield of H-2 production by fermentative bacteria by maximizing the number of electrons that are available for H-2. Here, we engineered Vitreoscilla hemoglobin (VHb) in Escherichia cob to study the effects of this versatile oxygen (O-2)-binding protein on oxic H-2 production in a closed batch system that was supplemented with glucose. The H-2 yields that were obtained with the VHb-expressing E. coil were greatly enhanced in comparison to the negative control cells in culture that started with high O-2 tensions. The formate hydrogen lyase (FHL) activity of oxically cultured, VHb-expressing cells was also much higher than that of the negative control cells. Through inhibitor studies and time-course experiments, VHb was shown to contribute to the improved H-2 yield primarily by increasing the efficiency of cellular metabolism during the aerobic phase before the onset of H-2 production and not by working as an O-2-scavenger during H-2 production. This new approach allowed more substrate to remain to be further utilized for the production of more H-2 from limited resources. We expect that VHb can be successfully engineered in potential aerobic H-2-producing microbial systems to enhance the overall H-2 production yield. In addition, the remarkably high FHL activity of oxically grown, VHb-expressing cells may make this engineered strain an attractive whole-cell biocatalyst for converting formate to H-2. Copyright (C) 2014, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved,